Down-regulation and silencing of allergen genes in transgenic peanut seeds

ABSTRACT

An allergen-free transgenic peanut seed is produced by recombinant methods. Peanut plants are transformed with multiple copies of each of the allergen genes, or fragments thereof, to suppress gene expression and allergen protein production. Alternatively, peanut plants are transformed with peanut allergen antisense genes introduced into the peanut genome as antisense fragments, sense fragments, or combinations of both antisense and sense fragments. Peanut transgenes are under the control of the 35S promoter, or the promoter of the Ara h2 gene to produce antisense RNAs, sense RNAs, and double-stranded RNAs for suppressing allergen protein production in peanut plants. A full length genomic clone for allergen Ara h2 is isolated and sequenced. The ORF is 622 nucleotides long. The predicted encoded protein is 207 amino acids long and includes a putative transit peptide of 21 residues. One polyadenilation signal is identified at position 951. Six additional stop codons are observed. A promoter region was revealed containing a putative TATA box located at position −72. Homologous regions were identified between Ara h2, h6, and h7, and between Ara h3 and h4, and between Ara h1P41B and Ara h1P17. The homologous regions will be used for the screening of peanut genomic library to isolate all peanut allergen genes and for down-regulation and silencing of multiple peanut allergen genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/958,324, filed on Oct. 6, 2004, which is currently pending. U.S.application Ser. No. 10/958,324 is a divisional application under 35U.S.C. §121 of U.S. application Ser. No. 09/715,036, filed on Nov. 20,2000, which is now issued as U.S. Pat. No. 6,943,010. U.S. applicationSer. No. 09/715,036 cites the benefit of U.S. Provisional PatentApplication 60/167,255, filed Nov. 19, 1999, which is now expired. Allof the foregoing U.S. patents and U.S. patent applications areincorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT FUNDING

This invention was made with Government support under Grant No. 96-02658awarded by the United States Department of Agriculture CooperativeStates Research Education and Extension Services (USDA/CSREES) CapacityBuilding Program. The Government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to transgenic peanut cells, peanut seeds andpeanut products with reduced or undetectable quantities of one or morepeanut allergen proteins. The invention also relates to isolated DNAsequences coding for peanut allergen proteins, as well as antisense,genes corresponding to each of the allergen protein genes. Furthermore,the invention relates to recombinant methods of reducing, andeliminating one or more of the peanut allergens in peanut seeds.

BACKGROUND OF THE INVENTION

Food allergy is a serious health problem, and can be life threatening.Public awareness of food allergies is at an all-time high, in part dueto the fact that allergic reactions to foods are being reported morefrequently. Up to 160 foods have been found to cause allergic reactions(Hefle, 1996, Crit. Rev. Food Sci. Nutr. 36:69-89).

The most common allergen-containing foods are peanuts, soybeans, treenuts, cow's milk, eggs, crustacea, and fish (Taylor, 1992, FoodTechnol., 148-152; Sampson, 1992. Food Technol., 141-144; and Burks,1992, Food Allergy News, 2:(1) 1). The frequency of food allergy ishighest in infancy and early childhood, and decreases with increasingage (Collin-Williams and Levy, 1984, Allergy to food other than milk. InFood intolerance, R. K. Chandra, ed., pp. 137-186. Elsevier, N.Y.).About 5% of children younger than three and 1.5% of the generalpopulation experience food allergy disorders, or about 4 millionAmericans suffer from food allergies (Sampson, 1997, JAMA 1997, 278(22): 1888-1894).

Food allergies are increasing worldwide, and peanut is one of the mostallergenic food products. It is estimated that over 600,000 children inthe United States have peanut allergies. While childhood allergies toegg and cow's milk may disappear, allergies to nuts, peanuts, soybeans,fish and shellfish tend to persist for the lifetime of the individual(Bock, 1982. J. Allergy Clin. Immunol., 69:173-177; Collin-Williams andLevy, 1984, supra).

Hypersensitive responses to peanut allergens can be fatal. Contact withthe slightest amount of peanut protein can be life threatening toparticularly sensitive individuals. There is little data on theincidence of near-fatal and fatal allergic reactions to food, but peanuthas been documented as a top offender (Taylor, 1987, NutritionalToxicol. 2: 173-177; Yunginger et al., 1988, JAMA 260:(10) 1450-1452;Evans et al., 1988, CMAJ, 139: (8) 231-232.; Burks et al. 1992; Bock1992, J. Allergy Clin. Immunol., 90:683-685). It is reported thatapproximately 125 people die each year in the USA of food-inducedanaphylaxis (Burks et al., 1999, Arch Allergy Immunol, 119 (3):165-172.)

The allergy can show up at the first exposure to peanuts, often beforethe age of three. Most people develop peanut allergies early in life,and few ever grow out of peanut allergies, even in adulthood. Allergicreactions to peanuts are often acute and severe (Sampson, 1990. Peanutanaphylaxis. J. Allergy Clin. Immunol. 86:1-3). The most commonmanifestation of peanut allergy is acute hives (or urticaria) followingexposure. However, some patients may rapidly develop severe angiodema,swelling of the face, bronchospasm and anaphylaxis, following exposure.Some individuals are so sensitive that they will develop symptoms ifthey kiss someone who has eaten peanuts or if they eat out of a foodutensil that has been in contact with peanuts.

Peanut (Arachis hypogaea), a crop grown worldwide, is an annual plantbelonging to the family Leguminosae, native to South America, and iscommercially grown in the southeastern regions of the United States,specifically in Alabama, Florida, Georgia, North Carolina, and Virginia,and in many other countries of the world. In the United States, severaltypes are grown, although the three most popular peanut types are theVirginia, Spanish, and runner varieties. Virginia peanuts are usedprimarily for whole kernel consumption and confections. Runner types areused most frequently for oil production and peanut butter (Woodroof,1983. In: Peanuts: Production, Processing, Products, Woodroof, Ed.,Westport, Conn.). Most of the peanut crop in the United States is usedfor the production of peanut butter. The most widely cultivated peanutcultivars in the USA are ‘Florunner’, ‘New Mexico Valencia’, ‘GeorgiaGreen’, and ‘Georgia Red’.

Although information about the nature and identity of allergeniccomponents of foods is quite limited, it is known that food allergensare most often proteins (Nordlee, et al., 1981., J. Allergy Clin.Immunol., 68:376-383), and provoke an abnormal immunoglobulinE-(IgE)mediated immunological reaction. Several allergenic peanutproteins have been isolated, identified, characterized and classified asminor or major allergens. (Burks A W, Williams L W, Helm R M,Connaughton C, Cockrell G, O'Brien T., J Allergy Clin Immunol 1991;88:172-179; Burks A W, Cockrell G, Connaughton C, Helm R M.; J AllergyClin Immunol 1994; 93:743-750; Gleeson P A, Jermyn M A., J Plant Physiol1977; 4,25; Kleber-Janke T, Crameri R, Appenzeller U, Becker W M,Schlaak M., Int Arch Allergy Immunol 1999; 119:265-274; Rabjohn P, HelmE M, Stanley J, West C M, Sampson H, Burks A W, Bannon G A., J ClinInvest 1999; 103(4):535-542; Sachs M I, Jones R T, Yunginger J W., JAllergy Clin Immunol 1981; 67(1):27-34; Stanley J S.,www.ncbi.nlm.nih.gov/htbin . . . ery?uid=1236995, 1996)

These proteins include glycoproteins, arachin, conarachin, peanutagglutinin and peanut phospholipase. Of these peanut protein allergens,six were classified as major allergens, with an estimated molecularweights of 44, 40, 33, 21, 20, and 18 kDa (De Jong et al., 1998, Clin.Exp. Allergy, 28: 743-751).

Burks et al. 1992 (J. Allergy Clin. Immunol. 90: 962-969) identified twomajor peanut allergens, designated Ara h 1 and Ara h 2, which areglycoproteins with isoelectric points and molecular weights of 4.55 and63,500 Daltons and 5.2 and 17,000 Daltons, respectively. These peanutallergens are stable at a temperature of up to 100° C., at pH conditionsbetween pH 2.8 and pH 10, and resistant to digestion by acid anddigestive enzymes. Peanut, peanut butter, and peanut flour retain theirallergenicity through processing, and crude peanut oil may also becontaminated with these proteins.

The allergens Ara h1 and Ara h2 are found in the cotyledon of peanut,and both are recognized by more than 90% of peanut-sensitive patients,establishing them as major allergens. Ara h1 has been isolated and acDNA clone produced and sequenced, making this the first peanut allergento be sequenced (Burks, et al., 1996, J. Clin. Invest. 96, 1715-1721).The partial cDNA sequences of Ara h2 (Stanley et al., 1997, Arch.Biochem. and Biophys, 342:244-253), Ara h3 (Rabjohn, et al., 1999, J.Clin. Invest. 103:535-542), Ara h4, Ara h6, and Ara h7 (Kleber-Janke, etal., 1999, Int. Arch. Allergy Immunol., 119:265-274) have also beenrecently cloned and sequenced.

Currently, no treatment exists for food allergies. Administration ofepinephrine and antihistamines is used to reverse the symptoms offood-allergic reactions. Thus, the most effective management strategy inthe prevention of peanut allergies is complete avoidance ofpeanut-containing foods (Schmidl, et al., 1994, Food Technol. 10:77-85).However, this is difficult to do, as it requires diligent reading oflabels and ingredient listings.

The peanut is a popular and important food, and provides a cheap sourceof protein and oil for human and animal consumption. Peanuts provideniacin, magnesium, Vitamin C, manganese and chromium in significantamounts and smaller amounts of potassium, Vitamin B6, folic acid,phosphorus, copper and biotin. Furthermore, peanut is widely used inboth western and oriental cooking, and is added to a variety of foodssuch as pastries, sandwiches, egg rolls, chili, syrups, flours, sauces,and confections (Nordlee et al., 1981; Yunginger et al., 1988; Evans etal., 1988; Burks et al., 1991). Because dining out is prevalent in thecurrent American lifestyle, the social stigma associated with refrainingfrom taking part in restaurant or party meals by allergic individualsbecause of the potential threat for accidentally ingesting peanut, makesthe strict avoidance of peanut unlikely and unrealistic (Heiner andNavin, 1975, J. Allergy Clin. Immunology 55:82). For example, one ofBritain's most promising young athletes died in June 1999, aftersuffering a seizure triggered by an accidental ingestion of peanut whileeating a chicken sandwich (The Independent-London-Jun. 21, 1999).

An investigation of a wide variety of commercially grown peanuts showedno naturally occurring allergen-free peanut lines. (Dodo H W, Marsic D,Mallender M, Cebert E, Viquez O M. Submitted to J. Allergy Clin.Immunology, 2000).

Therefore, there is a need for an alternative solution for the allergicindividual. Specifically, there is a need for allergen-free peanutplants, peanuts, and peanut products.

There is also a need for purified peanut allergen proteins which willenable the production of allergen-specific antibodies for detection ofallergen in food products, and for prophylaxis and treatment of allergicreactions to peanut.

Modern tools of molecular biology have the potential to offer newtransgenic allergen-free peanuts to the peanut-allergic population andthe peanut industry. Therefore, an understanding of the molecularstructure and regulatory features of the genes is needed to provideneeded information for gene silencing and production of allergen-freepeanut seeds.

SUMMARY OF THE INVENTION

The present invention provides an isolated polynucleotide moleculecomprising a peanut allergen antisense gene, and/or sense gene, orfragment thereof, operably linked to a promoter and a terminator, thepromoter and terminator functioning in a peanut cell. In particular,there is provided an isolated polynucleotide molecule comprising the Arah2 peanut allergen antisense gene together with its structural andregulatory features. Furthermore, there is provided a polynucleotidemolecule comprising an antisense gene that codes for an RNA moleculethat is complementary to, the mRNA molecule coded for by a peanutallergen protein gene selected from the group consisting of Ara h1, Arah2, Ara h3, Ara h4, Ara h5, Ara h6, and Ara h7, and any other peanutallergen gene that can induce an allergic reaction in humans.

The present invention further provides the isolated nucleotide sequencesof the antisense genes. Seed-preferred promoters, particularly aconstitutive promoter, an inducible promoter and a tissue-preferredpromoter, are operably linked to the antisense genes, and/or sensegenes.

The invention also provides modified transformation vectors such aspCB13, pB1426, pBI436, comprising a polynucleotide molecule havingpeanut allergen antisense genes, and/or a sense genes, or fragmentsthereof, operably linked to a promoter and a terminator, the promoterand terminator functioning in a peanut cell.

Yet further provided is a transformed bacterium containing apolynucleotide molecule comprising a peanut allergen antisense gene,and/or a sense gene or fragments thereof, operably linked to a promoterand a terminator, the promoter and terminator functioning in a peanutcell.

The invention also provides a peanut plant cell containing apolynucleotide molecule comprising a peanut allergen antisense gene,and/or a sense gene, or fragment thereof, operably linked to a promoterand a terminator, the promoter and terminator functioning in a peanutcell. Still further provided is a peanut plant containing a cellcomprising the polynucleotide molecule having a peanut allergenantisense gene, and/or a sense gene, or fragment thereof, operablylinked to a promoter and a terminator, the promoter and terminatorfunctioning in a peanut cell. Yet further, the invention provides a seedproduced by the peanut plant containing a cell comprising thepolynucleotide molecule having a peanut allergen antisense gene, and/ora sense gene, or fragment thereof, operably linked to a promoter and aterminator, the promoter and terminator functioning in a peanut cell.

The present invention provides methods for producing a transgenic peanutplant with reduced or undetectable allergen protein content in the seed,comprising transforming a recipient peanut plant cell with a DNAconstruct comprising a peanut allergen antisense gene, and/or a sensegene, or fragment thereof, regenerating a peanut plant from therecipient cell which has been transformed with the DNA construct, andidentifying a fertile transgenic peanut that produces seeds havingreduced or undetectable or undetectable allergen protein content. Therecipient peanut plant cell may be transformed by biolistic orAgrobacterium-mediated methods.

There is also provided a method wherein the recipient peanut plant cellis transformed with a DNA construct comprising an antisense gene, and/ora sense gene, having a nucleotide sequence based on the Ara h1, Ara h2,Ara h3, Ara h4, Ara h6, or Ara h7 genes, or any other allergen genes.Yet further is provided a method wherein the recipient peanut cell istransformed with a DNA construct comprising more than one antisensegene, and/or sense genes.

The invention further provides a method for producing a transgenicpeanut plant with reduced or undetectable allergen protein content inthe seed, comprising transforming a recipient peanut plant cell with aDNA construct comprising a peanut allergen gene, or fragment thereof,regenerating a peanut plant from the recipient cell which has beentransformed the DNA construct, and identifying a fertile transgenicpeanut that produces seeds having reduced or undetectable allergenprotein content. Still further, the invention provides a method whereinthe recipient peanut plant cell is transformed with the polynucleotideby the biolistic method. Yet further, the invention provides a methodwherein the recipient peanut plant cell is transformed with thepolynucleotide by the Agrobacterium-mediated method.

Also provided is a method wherein the recipient peanut plant cell istransformed with a DNA construct comprising a peanut allergen gene, orfragment thereof, that is the Ara h1, Ara h2, Ara h3, Ara h4, Ara h5,Ara h6, Ara h7 or any other peanut allergen gene. The invention furtherprovides a method wherein the recipient peanut cell is transformed witha DNA construct comprising more than one peanut allergen gene.

Also provided is a method wherein homologous sequence region between twoor more peanut allergen genes is used to down-regulate peanut allergens.A method is provided for producing a transgenic peanut plant withreduced or undetectable allergen protein content in the seed comprisingthe steps of identifying a homologous region common to more than one Arah allergen gene; cloning the homologous region in a vector wherein thehomologous region is operably linked to a promoter; transforming arecombinant peanut cell with the vector; and identifying a regeneratedfertile transgenic peanut plant that produces seed having reduced orundetectable allergen protein content.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Southern hybridization of (A) the Bam HI digestionpattern of the positive 50 kb lambda clone for Ara h2 gene (lane 3),Lambda DNA/Hind III markers (lane 1), 1 kb DNA step ladder (lane 2); (B)hybridization of an 80-mer labeled probe with a subcloned 12 kb BamHI-fragment; and (C) hybridization of an 62-mer labeled probe with asubcloned 6.5 Bam HI-fragment (clones 1-6).

FIG. 2 shows the nucleotide and deduced amino acid sequences (SEQ ID NOS1-2, respectively) of peanut allergen Ara h2 gene. The figure also showsa putative TATA box, an ATG initiation codon, the first stop codon(TGA), and putative polyadenylation signal (bold). Six additional stopcodons are underlined. The deduced polypeptide encoded by the openreading frame has 207 amino acids residues and includes a putativesignal peptide of 21 amino acid residues (underlined).

FIG. 3 shows the PCR amplified region (in capital letters) of Ara h2genomic DNA (SEQ ID NO: 3), cloned in transformation vectors (pUC18 andpBI434) in sense and antisense orientations to down-regulate Ara h2, Arah6, and Ara h7 allergens in peanut. This region is a portion of thesequence homology region between Ara h2, Ara h6, and Ara h7 allergens.

FIG. 4 shows the PCR amplified region (in capital letters) of Ara h3cDNA (SEQ ID NO: 4), cloned in transformation vectors (pUC18 and pBI434)in sense and antisense orientations to down-regulate Ara h3, and Ara h4allergens in peanut. This region is a portion of the sequence homologyregion between Ara h3 and Ara h4 allergens.

FIG. 5 shows PCR amplified region (in capital letters) of Ara h1 P41BcDNA (SEQ ID NO: 5), cloned in transformation vectors (pUC18 and pBI434)in sense and antisense orientations to down-regulate Ara h1 P41 B, andAra h 1 P17 allergens in peanut. This region is a portion of thesequence homology region between Ara h1 P41B and Ara h1 P17 allergens.

FIG. 6 is a schematic representation of plasmid constructs todown-regulate peanut allergens in transgenic peanuts.

FIG. 7 shows the PCR amplified region of Ara h5 cDNA (SEQ ID NO: 6)(shown in bold), cloned in sense and antisense orientations intransformation vectors (pUC18 and pBI434), to down-regulate Ara h5allergen in peanut.

FIG. 8 shows diagrams of the plasmid constructs used in biolistic andAgrobacterium-mediated transformation of peanut.

FIG. 9 shows the nucleotide sequence (residues 1-154 of SEQ ID NO:1) ofthe Ara h2 promoter upstream of the ATG initiation codon.

DEFINITIONS

As used herein, the term gene should be understood to be a full-lengthDNA sequence encoding a protein or an RNA molecule, as well as atruncated fragment thereof A gene can be naturally occurring orsynthetic.

Marker gene should be understood as a gene encoding a selectable marker(e.g., encoding antibiotic resistance) or a screenable marker (e.g.,encoding a gene product which permits detection or transformed cells orplants). The marker gene for the polynucleotide molecule of the presentinvention can be any nucleotide sequence which codes for a protein orpolypeptide which allows transformed cells to be distinguished fromnon-transformed cells. The marker gene can be, for example, a herbicideresistance gene, an antibiotic resistance gene, a β-glucuronidase (GUS)gene, or a luciferase gene.

A promoter is a nucleotide sequence upstream from the transcriptionalinitiation site and which contains all the regulatory regions requiredfor transcription. Examples of promoters suitable for use in DNAconstructs of the present invention include viral, fungal, bacterial,animal and plant-derived promoters capable of functioning in plantcells. The promoter may be selected from so-called constitutivepromoters or inducible promoters. If a promoter is an induciblepromoter, then the rate of transcription increases in response to aninducing agent. In contrast, the rate of transcription is not regulatedor largely unregulated by an inducing agent, if the promoter is aconstitutive promoter.

Examples of suitable inducible or developmentally regulated promotersinclude the napin storage protein gene (induced during seeddevelopment), the malate synthase gene (induced during seedlinggermination), the small sub-unit RUBISCO gene (induced in photosynthetictissue in response to light), the patatin gene (highly expressed inpotato tubers) and the like. Examples of suitable constitutive promotersinclude the cauliflower mosaic virus 35S (CaMV 35S) and 19S (CaMV 19S)promoters, the nopaline synthase promoter, octopine synthase promoter,heat shock 80 (hsp 80) promoter and the like. It will be appreciatedthat the promoter employed in the present invention should be strongenough to control the transcription of a sufficient amount of anantisense RNA molecule to cause an inhibition of expression of a peanutallergen in transformed cells.

A tissue-preferred promoter is a DNA sequence that, when operably linkedto a gene, directs a higher level of transcription of that gene in aspecific tissue than in some or all other tissues in an organism.Examples of such promoters are a stem-specific promoter such as theAdoMet-synthetase promoter (Peleman et al., 1989, The Plant Cell1:81-93), a tuber-specific promoter (Rocha-Sosa et al., 1989, EMBO J.8:23-29). For example, a seed-preferred promoter is a DNA sequence thatdirects a higher level of transcription of an associated gene in plantseeds. Examples of seed-preferred promoters include the seed specificpromoter of the USP gene of Vicia faber (U.S. Pat. No. 5,917,127); the7S protein promoter of soybean (Bray et al., 1987, Planta 172:364-370)and the 2S promoter (Krebbers et al., 1988, Plant Physiol. 87:859-866).

A terminator is a DNA sequence at the end of a transcribed unit whichsignals termination of transcription. These elements are3′-non-transcribed sequences containing polyadenylation signals whichact to cause the addition of polyadenylate sequences to the 3′ end ofprimary transcripts. Examples of terminators particularly suitable foruse in nucleotide sequences and DNA constructs of the invention includethe nopaline synthase polyadenylation signal of Agrobacteriumtumefaciens, the 35S polyadenylation signal of CaMV, octopine synthasepolyadenylation signal and the zein polyadenylation signal from Zeamays.

An isolated nucleic acid molecule is a fragment of nucleic acid moleculethat has been separated from the nucleic acid of an organism or othernatural environment of the nucleic acid an isolated nucleic acidmolecule includes a chemically-synthesized nucleic acid molecule. Otherexamples of isolated nucleic acid molecule include in vivo or in vitrotranscripts of the nucleic acids of the present invention.

Isolated polypeptides are polypeptides not in their naturally occurringform or have been purified to remove at least some portion of cellularor non-cellular molecules with which the proteins are naturallyassociated. However, the “isolated” protein may be included incompositions containing other polypeptides for specific purposes, forexample, as stabilizers, where the other polypeptides may occurnaturally associated with at least one polypeptide of the presentinvention.

The terms “complementary” or “complementarity” refer to the capacity ofpurine and pyrimidine nucleotides to associate non-covalently to formpartial or complete double stranded nucleic acid molecules. Thefollowing base pairs are naturally complementary: guanine (G) andcytosine (C); adenine (A) and thymine (T); and adenine (A) and uracil(U).

Complementary DNA (cDNA) is a single-stranded DNA molecule that isformed from a mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of an mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

The term expression refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides. In the case of an antisense gene,expression involves transcription of the antisense DNA into an antisenseRNA molecule that is complementary to the sense mRNA.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A DNA molecule can be designed tocontain an RNA polymerase II template in which the RNA transcript has asequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an antisense RNA and a DNA sequence that codes forthe antisense RNA is termed an antisense gene. An antisense RNA moleculeinhibits the expression of the gene to which it corresponds.

A vector is a DNA molecule, such as a plasmid, cosmid, viruses orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain a marker gene and one or asmall number of restriction endonuclease recognition sites for insertionof foreign DNA sequences without affecting the essential biologicalfunction of the vector.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” or “operatively linkedto” the regulatory elements.

“Host cell” refers to any eukaryotic, prokaryotic, or other cell that issuitable for propagating or expressing an isolated nucleic acid that isintroduced into the cell by any suitable means known in the art. Thecell can be part of a tissue or organism, isolated in culture or in anyother suitable form. A recombinant host may be any prokaryotic oreukaryotic cell that contains either a cloning vector or expressionvector. This term also includes those prokaryotic or eukaryotic cellsthat have been genetically engineered to contain an isolated gene in thechromosome or genome of the host cell.

A transgenic peanut plant is a plant having one or more plant cells thatcontain a foreign gene. The foreign gene is usually non-native, meaningthat it is originated from a source other than the host plant and doesnot share sequence homology to the host genome. The foreign gene mayalso be native, meaning that it has the nucleotide sequence found in thehost. The transgenic plant is made by one of many transformation methodswell-known in the art. As used herein, a fertile transgenic plant iscapable of transmitting a foreign gene to its progeny of furtherdescendants. As used herein, the term transformation refers toalteration of the genotype of a host plant by the introduction of nativeor non-native nucleic acid sequences into the genomes of the plant cell.

Peanut allergen variants, according to the invention, include DNA orprotein molecules that resemble, structurally and functionally, thepolynucleotide with the sequence of any peanut allergen gene. Peanutallergen genes that can be used for the present invention include Arah1, Ara h2, Ara h3, Ara h4 Ara h5, Ara h6, Ara h7, and any other suchgenes that are identified and cloned which induce an allergic responsein a human.

Hybridizing peanut allergen variants: Nucleic Acid variants within theinvention also may be described by reference to their physicalproperties in hybridization. One skilled in the field will recognizethat nucleic acid can be used to identify its complement or homologue,using nucleic acid hybridization techniques. It will also be recognizedthat hybridization can occur with less than 100% complementarity.However, given appropriate choice of conditions, hybridizationtechniques can be used to differentiate among DNA sequences based ontheir structural relatedness to a particular probe. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y.

Structural relatedness between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. Stringent conditionsstrongly disfavor hybridization, and only the most structurally relatedmolecules will hybridize to one another under such conditions.Conversely, non-stringent conditions favor hybridization of moleculesdisplaying a lesser degree of structural relatedness. Hybridizationstringency, therefore, directly correlates with the structuralrelationships of two nucleic acid sequences (Bolton et al., 1962, Proc.Natl. Acad. Sci. 48:1390.

Hybridization stringency is thus a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents that disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding.

Hybridization usually is done in two stages. First, in the “binding”stage, the probe is bound to the target under conditions favoringhybridization. A representative hybridization solution comprises 6×SSC,0.5% SDS, 5×Denhardt's solution and 100 μg of non-specific carrier DNA.See Ausubel et al., supra, section 2.9, supplement 27 (1994). A stock20×SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH7.0. Of course many different, yet functionally equivalent, bufferconditions are known. For high stringency, the temperature is betweenabout 65° C. and 70° C. in a hybridization solution of 6×SSC, 0.5% SDS,5×Denhardt's solution and 100 μg of non-specific carrier DNA. Moderatestringency is between at least about 40° C. to less than about 65° C. inthe same hybridization solution. In both cases, the preferred probe is100 bases.

Second, the excess probe is removed by washing, which is most importantin determining relatedness via hybridization. Washing solutionstypically contain lower salt concentrations. A medium stringency washsolution contains the equivalent in ionic strength of 2×SSC and 0.5-0.1%SDS. A high stringency wash solution contains the equivalent in ionicstrength of less than about 0.2×SSC and 0.1% SDS, with a preferredstringent solution containing about 0.1×SSC and 0.1% SDS. Thetemperatures associated with various stringencies are the same asdiscussed above for “binding.” The washing solution also typically isreplaced a number of times during washing. For example, typical highstringency washing conditions comprise washing with 2×SSC plus 0.05% SDSfive times at room temperature, and then washing with 0.1×SSC plus 0.1%SDS at 68° C. for 1 h. Blots containing the hybridized, labeled probeare exposed to film for one to three days.

Particularly preferred molecules are at least 75% of the length of thosemolecules.

Structural variants may also be due to substitutions, insertions,additions, and deletions. With regard to amino acid sequence,“substitutions” generally refer to alterations in the amino acidsequence that do not change the overall length of the polypeptide, butonly alter one or more amino acid residues, substituting one for anotherin the common sense of the word. “Insertions,” unlike substitutions,alter the overall length of the polypeptide. Insertions add extra aminoacids to the interior (not the amino- or carboxyl-terminal ends) of thesubject polypeptide. “Deletions” diminish the overall size of thepolypeptide by removal of amino acids from the interior or either end ofthe polypeptide. Preferred deletions remove less than about 30% of thesize of the subject molecule. “Additions,” like insertions, also add tothe overall size of the protein. However, instead of being made withinthe molecule, they are made on the N- or C-terminus of the encodedprotein. Unlike deletions, additions are not very size-dependent.Indeed, additions may be of virtually any size. Preferred additions,however, do not exceed about 100% of the size of the native molecule.The artisan understands “additions” also to encompass adducts to theamino acids of the native molecule.

In general, both the DNA and protein molecules of the invention can bedefined with reference to sequence identity. As used herein, “sequenceidentity” refers to a comparison made between two molecules usingstandard algorithms well-known in the art. Although any sequencealgorithm can be used to define “sequence identity,” for clarity, thepresent invention defines identity with reference to the Smith-Watermanalgorithm, where the open reading frame of a gene is used as thereference sequence to define the percentage identity of polynucleotidehomologues over its length. When “sequence identity” is used withreference to a polypeptide, the entire polypeptide having the sequenceof a polypeptide of interest is used as a reference sequence todetermine the percent identity of polypeptide homologues over itslength.

Preferred polynucleotides are those having at least about 80% sequenceidentity to the open reading frame. Particularly preferredpolynucleotides have at least about 90% sequence identity. Even morepreferred polynucleotides have at least about 95% sequence identity, andmost preferred polynucleotides have at least 98% sequence identity. Asused herein, two nucleic acid molecules or proteins are said to “sharesignificant sequence identity” if the two contain regions that possessgreater than 85% sequence (amino acid or nucleic acid) identity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 1. Overview

The present invention discloses the isolated, sequenced andcharacterized genomic clone of the major peanut allergen gene Ara h 2.The present invention also provides an isolated polynucleotide moleculecomprising the coding sequence for each of the peanut allergen genesoperably linked to a promoter and a terminator, the promoter andterminator functioning in a peanut cell. The isolated peanut allergengene, or fragment thereof, is operably linked to a selected promoter andtransformed into peanut cells to make a stably transformed plant.

Peanut seeds comprising multiple copies of a peanut allergen geneexhibit reduced or undetectable allergen protein content due tocosuppression, antisense RNAs, or double-stranded RNAs by combiningsense and antisense genes. The selected promoter may be a constitutiveor tissue-preferred promoter such as a seed-preferred promoter. Peanutplants may be transformed with more than one peanut allergen gene, orfragment of each gene, in order to produce peanut seeds containingreduced or undetectable quantities of more than one peanut allergenproteins. Alternatively, peanut plants may be transformed with a DNAconstruct comprising more than one peanut allergen gene, or fragment ofeach gene, in order to produce peanut plants and seeds containingreduced or undetectable quantities of more than one peanut allergenproteins.

Furthermore, the peanut plants may be transformed with a DNA constructcomprising one or more polynucleotide sequences found in more than onepeanut allergen gene in a process to produce peanut plants and seedscontaining reduced or undetectable quantities of several differentpeanut allergen proteins. In a preferred embodiment, the peanut allergengene is selected from the group consisting of Ara h1, Ara h2, Ara h3,Ara h4, Ara h5, Ara h6, and Ara h7, and any other peanut allergen gene,and fragments thereof.

The invention also provides an isolated polynucleotide moleculecomprising a peanut allergen antisense gene, and/or a sense gene, and/orcombined antisense and sense genes, operably linked to a promoter and aterminator, the promoter and terminator functioning in a peanut cell.The isolated peanut allergen antisense gene, and/or sense gene, and/orcombined antisense and sense genes, or fragment thereof, is operablylinked to a selected promoter and transformed into peanut cells to makea stably transformed plant. Peanut seeds comprising a peanut allergengene exhibit reduced or undetectable allergen protein content. Theselected promoter may be a constitutive or tissue-preferred promotersuch as a seed-preferred promoter.

Peanut plants may be transformed with more than one peanut allergenantisense gene, and/or sense gene, and/or combined antisense and sensegenes, or fragment of each gene, in order to produce peanut plants andseeds containing reduced or undetectable quantities of several differentpeanut allergen proteins. Alternatively, peanut plants may betransformed with a polynucleotide comprising more than one peanutallergen antisense genes, or fragments thereof, in a process to producepeanut plants and seeds containing reduced or undetectable quantities ofseveral different peanut allergen proteins. Furthermore, the peanutplants may be transformed with a DNA construct comprising one or moreantisense genes comprising a polynucleotide sequence that iscomplementary to a DNA sequence found in more than one peanut allergengene in a process to produce peanut plants and seeds containing reducedor undetectable quantities of several different peanut allergenproteins.

Peanut plants may be transformed with a DNA construct comprising one ormore sense genes, comprising a polynucleotide sequence that is similarto a DNA sequence found in more than one peanut allergen genes in aprocess to produce peanut plants and seeds containing reduced orundetectable quantities of several different peanut allergen proteins.

In a preferred embodiment, the peanut allergen antisense gene generatesan RNA molecule which is complementary to a sense mRNA molecule encodinga peanut major allergen protein selected from the group consisting ofAra h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, and any otherallergen gene, and fragments thereof. The peanut allergen sense genegenerates an RNA molecule which is identical to a sense mRNA moleculeencoding a peanut major allergen protein selected from the groupconsisting of Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7,and any other allergen gene, and fragments thereof. A combination of apeanut allergen antisense gene and sense gene generates a simultaneousexpression of sense and antisense sequences corresponding to a peanutmajor allergen protein selected from the group consisting of Ara h1, Arah2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, and any other allergen gene,and fragments thereof.

Also provided is a vector, a bacterium, and a peanut plant cellcomprising the polynucleotide molecules of the invention. Still furtherprovided is a method for producing a transgenic peanut plant withreduced or undetectable allergen content. The method comprises a)preparing the polynucleotide molecules of the instant invention; b)transforming a recipient peanut plant cell with the polynucleotidemolecules of the instant invention; c) regenerating a peanut plant fromthe recipient cell which has been transformed with the polynucleotidemolecule; and d) identifying a fertile, transgenic peanut plantcomprising the polynucleotide molecule and reduced or undetectableallergen content. A preferred embodiment of the method utilizes abiolistic apparatus or a Agrobacterium Ti plasmid for the transformationof the peanut plant cell. The polynucleotide molecules of the instantinvention include peanut allergen genes, peanut allergen antisensegenes, peanut allergen sense genes, and a combination of peanut allergenantisense and sense genes, and fragments thereof.

The present invention also provides methods for testing for allergens intransgenic peanuts using ELIZA.

The present invention also provides methods, utilizing traditional plantbreeding procedures, for incorporating the allergen-free peanutgermplasm into diverse peanut genetic backgrounds.

2. Peanut Allergen Genes

Peanut contains multiple allergens. An allergen is defined as a moleculethat elicits an abnormal immunoglobulin E (IgE)-mediated immunologicalreaction within certain individuals. Burks et al., 1992, J. AllergyClin. Immunol., 90: 962-969, identified two glycoproteins which aremajor peanut allergens, Ara h1 and Ara h2, with molecular weight andisoelectric points of 63.5 kDa and 4.55 and 17 kDa and 5.2 respectively.De Jong et al., 1998 Clin. Exp. Allergy, 28 (6): 743-751, identified andclassified six peanut proteins as major allergens with an estimatedmolecular weight of 44, 40, 33, 21, 20, and 18 kDa. Rabjohn et al.,1999, J. Clin. Invest. 103(4): 535-542, isolated another peanut allergenAra h3 Kleber-Janke et al., 1999, Int. Arch. Allergy Immunol.119:265-274, identified and cloned Ara h4, Ara h5, Ara h5, Ara h6 andAra h7 by Kleber-Janke T., et al., 1999.

The nucleotide sequences of the published Ara clones can be obtained asfollows: Arah1, Clone P41B (GenBank Accession number L34402), Burks W,Cockrell, Stanley S T, Helm R M and Bannon G A (1995), Clin. Invest 96:1715-1721; Arah1 Clone P17 (GenBank Accession number L38853), Burks W,Cockrell, Stanley S T, Helm R M and Bannon, Unpublished; Arah2 cDNA(GenBank Accession number L7797), Stanley J S, Unpublished; Arah2genomic DNA, Viquez O M, Summer C G and DODO W H (2000), accepted forpublication in The Journal of Allergy and Clinical Immunology; Arah3cDNA (GenBank Accession number AF093541), Robinson P, Helm E M, StanleyS J, West C M, Sampson H A, Burk A W and Banonn G A (1998) Unpublshed;Arah4 cDNA (GenBank Accession number AF086821), Kleber-Janke T, CrameriR, Appenzeller U, Schlaak M, and Becker W M (1999), Int Arch. AllergyImmunol 119 (4) 265-274; Arah5 cDNA (GenBank Accession number AF059616),Kleber-Janke T, Crameri R, Appenzeller U, Schlaak M, and Becker W M(1999), Int Arch. Allergy Immunol 119 (4) 265-274; Arah6 cDNA (GenBankAccession number AF092846), Kleber-Janke T, Crameri R, Appenzeller U,Schlaak M, and Becker W M (1999), Int Arch. Allergy Immunol 119 (4)265-274; Arah7 cDNA (GenBank Accession number AF091737), Kleber-Janke T,Crameri R, Appenzeller U, Schlaak M, and Becker W M (1999), Int Arch.Allergy Immunol 119 (4) 265-274.

3. Isolation of Genes Encoding Peanut Allergen Proteins

Several different methods are available for isolating genes coding forpeanut allergen proteins. Most approaches begin with the purification ofthe protein. The purified protein is then subjected to amino acidmicrosequencing, either directly or after limited cleavage. The partialamino acid sequence that is obtained can be used to design degenerateoligonucleotide probes or primers for use in the generation of unique,nondegenerate nucleotide sequences by polymerase chain reaction (PCR),sequences that can in turn be used as probes for screening genomic DNAlibraries. Antibodies raised against purified protein may also be usedto isolate DNA clones from expression libraries.

Alternatively, the sequences of DNA coding for related proteins may beused as starting points in a cloning strategy, so-called “cloning byhomology”. Another way of utilizing sequence information from differentspecies is to take advantage of shorter areas of high sequence homologyamong related DNAs from different species and to perform PCR to obtain“species-specific” nondegenerate nucleotide sequences. Such a sequencecan then be used for library screening or even for direct PCR-basedcloning. Detection of the desired DNA can also involve the use of PCRusing novel primers.

Libraries are screened with appropriate probes designed to identify thegenomic DNA of interest. For expression libraries (which express theprotein), suitable probes include monoclonal or polyclonal antibodiesthat recognize and specifically bind to the peanut allergen protein.Screening the genomic DNA library with the selected probe may beaccomplished as described in the example below.

Screening genomic DNA libraries using synthetic, degenerateoligonucleotides based on partial amino acid sequences, oroligonucleotides based on 5′ cDNA sequences, or oligonucleotides basedon homologous regions between several allergens of purified known peanutallergen proteins as probes, are the preferred methods of thisinvention.

The oligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous to minimize false positives. Thedesign of actual nucleotide sequence(s) of the probe(s) is based onregions of the peanut allergen protein that have the least codonredundancy. The oligonucleotides may be degenerate at one or morepositions, i.e., two or more different nucleotides may be incorporatedinto an oligonucleotide at a given position, resulting in multiplesynthetic oligonucleotides. The use of degenerate oligonucleotides is ofparticular importance where a library is screened from a species inwhich preferential codon usage is not known.

The oligonucleotide can be labeled according to procedures well known inthe art, such that it can be detected upon hybridization to DNA in thelibrary being screened. A preferred method of labeling is to use ATP andpolynucleotide kinase to radiolabel the 5′ end of the oligonucleotide.However, other methods may be used to label the oligonucleotide,including, but not limited to, biotinylation or enzyme labeling.

4. Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, (c)purification techniques, or combinations thereof, as are well known tothose skilled in the art. The nucleic acids may conveniently comprisesequences in addition to a polynucleotide of the present invention. Forexample, a multi-cloning site comprising one or more endonucleaserestriction sites may be inserted into the nucleic acid to aid inisolation of the polynucleotide. Also, translatable sequences may beinserted to aid in the isolation of the translated polynucleotide of thepresent invention. For example, a hexa-histidine marker sequenceprovides a convenient means to purify the proteins of the presentinvention. Additional sequences that may be inserted include adapters orlinkers for cloning and/or expression. Use of cloning vectors,expression vectors, adapters, and linkers is equally well known to thoseskilled in the art.

The various restriction enzymes disclosed and described herein arecommercially and/or available and the manner of use of the enzymesincluding reaction conditions, cofactors, and other requirements foractivity are well known to one of ordinary skill in the art (New EnglandBioLabs, Boston; Life Technologies, Rockville, Md.). Reaction conditionsfor particular enzymes are preferably carried out according to themanufacturer's recommendation.

A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids using methods and reagents known in the art.

i) Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention can be obtainedfrom biological sources using any number of cloning methodologies knownto those of skill in the art. Oligonucleotide probes that selectivelyhybridize to the polynucleotides of the present invention may be used toidentify the desired sequence in a cDNA or genomic DNA library.Isolation of RNA and construction of cDNA and genomic libraries is wellknown to those of ordinary skill in the art.

ii) Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis using the solid phase phosphoramiditetriester method (Beaucage and Caruthers, Tetra. Letts. 22(20): 1859-1862(1981)); an automated synthesizer (VanDevanter et al., Nucleic AcidsRes., 12: 6159-6168 (1984)); or the solid support method of U.S. Pat.No. 4,458,066. Chemical synthesis generally produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill willrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

iii) Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a peanut allergen gene, peanut allergen antisense gene, or apeanut sense gene, or a combination of peanut allergen antisense andsense genes, or fragments thereof, operably linked to transcriptionalinitiation regulatory sequences that will direct the transcription ofthe polynucleotide in the intended host cell. Both heterologous andendogenous (native) promoters can be employed to direct expression.These promoters can also be used, for example, in recombinant expressioncassettes to drive expression of antisense, sense or a combination ofantisense and sense nucleic acids, to reduce or to eliminate peanutallergen content in a desired tissue.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution. Suitable promoters include the phage lambda PL promoter,the E. coli lac, trp and tac promoters, the SV40 early and latepromoters and promoters of retroviral LTRs, to name a few. Othersuitable promoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will preferably include a translation initiation (AUG) atthe beginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Such markers include, e.g.,dihydrofolate reductase or neomycin resistance for eukaryotic cellculture and tetracycline, ampicillin or kanamycin resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium cells; and fungalcells, such as yeast cells.

5. Control of Peanut Allergen Gene Expression

The present invention discloses methods to reduce or eliminate theexpression of peanut allergen genes on the basis of antisense,co-suppression, dsRNA technology, and ribozymes.

Plant transformation technologies utilize molecular strategies todown-regulate or to inhibit the expression of endogenous plant genes.These proven strategies have been used to make the allergen-free-peanutplants of the instant invention. They include the antisense RNAstrategy, homology dependent gene silencing (HDGS), and thedouble-stranded RNA method.

In the antisense RNA strategy (reviewed by Watson C F, and Don Grierson(1992), Antisense RNA in Transgenic Plants, fundamentals andapplications. Hiatt Ed. p 255-281), it is considered that an antisensetranscript suppresses gene expression post-transcriptionally byinhibiting RNA processing, transport from the nucleus, and translation,by hybridization with the sense molecules.

Matzke M A, and Matzke A T M (Plant Physiol. (1995), 107: 679-685),reviewed the mechanisms involved in homology dependent-gene silencing(HDGS). Double-stranded RNA (dsRNA) is a new tool to suppress geneexpression in a number of organisms (Fire et al., 1998 Nature391:806-811; Montgomery et al., 1998 Proc. Natl. Acad. Sci.95:15502-15507; Kennerdell and Carthew, 1998 Cell 95:1017-1026,Misquitta and Paterson, 1999, Proc. Natl. Acad. Sci. 96:1451-1426, Ngoet al., 1998, Proc. Natl. Acad. Sci. 95:14687-14692) including plants(Waterhouse et al., 1998, Proc. Natl. Acad. Sci. 95:13959-13964).Double-stranded RNA has a very high specificity in suppressing theexpression of the gene from which the dsRNA sequence is derived withoutdetectable effect on the expression of genes unrelated in sequence (Fireet al., 1998, Nature 391:806-811). The molecular mechanisms by whichdsRNA generates gene silencing are not well understood yet. It ishowever speculated that, the gene silencing is a result of a cellulardefense to dsRNA formation from nuclear transcripts.

Two classes of HDGS are distinguished by their effect on transcriptionon the target gene. Examples of transcriptional gene silencing (TGS) areknown in which the phenomenon of DNA methylation is the key factor. Thepromoter of the target gene is methylated, and thereby, becomesinactive. (Brusslan A J, Karlin-Neumann G A, Huang Lu and Tobin M E(1993), The Plant Cell 5: 667-677; Neuhuber F, Park Y D, Matzke A J M,Matzke M A, (1994) Molecular and General Genetics 244: 230-241) When atransgene integrates into a heavily methylated chromosomal region, it israpidly silenced. By DNA-DNA interactions, a transgene locus that issilenced can lead to silencing of homologous genes. When the silencedlocus is methylated the target locus also becomes methylated.

Double-stranded DNA blocks the activity of genes by artificiallyproviding sense and antisense RNA corresponding to the target gene. Genesilencing by dsRNA is a post-transcriptional process (PTGS). It isdemonstrated that triggering of PTGS by direct introduction of foreignRNA requires that both the sense and the antisense strands are providedexogenously, even if a cell already has substantial pool of naturallysynthesized sense and antisense RNAs from distinct chromosomal sites, toproduce a PTGS response (Ngo et al., 1998; Fire et al., 1998; Waterhouseet al., 1998;) All three strategies, antisense RNA, co-suppression anddouble-stranded RNA are used in the present invention.

Antisense technology is a versatile approach for controlling expressionof endogenous cellular genes and extinguishing cellular gene expression.The principle is to introduce into a cell an RNA or a single strandedDNA molecule complementary to the mRNA of the target gene (the“antisense molecule”). The antisense molecule can base-pair with thenaturally occurring corresponding cellular mRNA and prevent itstranslation. The protocol was originally developed for the control ofthe gene encoding polygalacturonase during fruit ripening in tomato(Smith et al. 1988, Nature 334:724-726). Considerable effort has beendevoted to the development of antisense RNA technology for theproduction of novel plant mutants which have the advantage of beingstably inherited (Schuch, 1991, Soc. Exp. Biol. 117-127).

Antisense technology, however, has not been applied to peanut. Prior tothis invention, there has not been peanut plants or germplasm, whethernaturally occurring or genetically engineered, that is partially orcompletely allergen free. In fact, in an ELISA screen of 32 commercialpeanut cultivars by the inventors of the instant invention, noallergen-free cultivar was identified, although a significant differencein allergen level was found among the cultivars.

The present invention provides a nucleotide sequence which is anantisense gene encoding an antisense RNA molecule which has a nucleotidesequence complementary to a sense mRNA molecule that codes for a majorpeanut allergen protein. This antisense gene is under transcriptionalcontrol of a promoter and a terminator, both promoter and terminatorcapable of functioning in peanut plant cells.

The antisense gene can be of any length provided that the antisense RNAmolecule encoded by the antisense gene is sufficiently long to form acomplex with a sense mRNA molecule encoding a peanut allergen protein.For the purposes of the description of the present invention, theantisense gene can be from about 50 nucleotides in length up to a lengthwhich is equivalent to the full length of the gene. Preferably, thelength of the DNA encoding the antisense RNA will be from 100 to 1500nucleotides. The preferred gene of the present invention is a DNA thatcodes for an RNA having substantial sequence identity or similarity tothe mRNA encoding a peanut allergen protein. Thus the antisense DNA ofthe present invention may be selected from the group of peanut allergengenes or fragments thereof.

The antisense, sense and the combination of antisense and sense peanutallergen genes may consist of a plurality of subsequences, wherein eachsubsequence codes for an antisense RNA molecule, a sense RNA moleculeand a dsRNA molecule directed to a different peanut allergen gene or adifferent portion of the same peanut allergen gene. Naturally, theskilled artisan will appreciate that the subsequences can be adjacent toone another, or noncontiguous, in any order. The invention also providesfor a nucleotide sequence that is a variant of the antisense genesdescribed herein.

The present invention discloses a DNA construct comprising thenucleotide sequence according to the invention, as well as a modifiedtransformation vectors comprising the sequence or construct. The vectormay be a plasmid or virus. The vector may be the Ti plasmid ofAgrobacterium tumefaciens. The vector advantageously carries aselectable marker gene. The nucleotide sequence of the invention maycode for an mRNA which comprises, in the 5′ to 3′ direction, (i) apromoter, (ii) at least one peanut allergen antisense gene, and (iii) aterminator. In the DNA construct shown in FIG. 6., the Arah2 gene isexpressed from its native promoter, and the marker genes are operablylinked to the CaMV35s promoter.

Conserved sequences from different allergen genes may be used todown-regulate all known and/or existing allergens (Arah1, Arah2, Arah3,Arah4, Arah5, Arah6, Arah7, and any other allergens) in peanut plants.Suppression of expression of more than one allergen gene may be done byintroducing multiple copies of a gene or gene fragment into a construct,using sense or antisense homologous regions. The resultant constructcontains more than one homologous antisense or sense gene fused in frameand may be used to reduce or eliminate expression of more than onetarget allergen gene.

Suitable transformation vectors such as pUC 18, pBI426 and modifiedversions of pBI426 (shown in FIG. 8) are used for carrying out biolistictransformation. Modified versions of pBI434 (Dalta et al., 1991), (alsoshown in FIG. 8), a binary vector for transformation using Agrobacteriumtumefaciens (FIG. 6). (See Example 3, below) Transformation vectorscarry the transgenes, flanked by a promoter such as the Arah2 promoter,or the 35S promoter, and the nopaline synthase terminator. The peanutallergen gene may be portions of the open reading frame (ORF) of peanutallergens Arah1, Arah2, Arah3, Arah4, Arah5, Arah6, Arah7, or any otherallergen gene.

Different types of transformation cassettes are made to down-regulatepeanut allergens. Regions of homology between the nucleotide sequencesof different allergens are PCR amplified from the genomic DNA of Arah2.The PCR product is cloned in both antisense and sense orientation intothe same transformation vector to produce dsRNA in transformed peanutcells. Or, the antisense construct is used in co-transformation with theconstruct to produce dsRNA in transformed peanut cells. Anotheralternative, is to synthesize at least 100 base pair oligonucleotidescorresponding to the homology region between the above three allergensto make the antisense and sense transgene constructs.

For control of allergen genes Arah3, and Arah4, it is noted that cDNAsequences of these two allergens have 95% homology, shown in capitalletters in the sequence in FIG. 4. A portion of two hundred base pairswithin the homology region is PCR amplified, and then cloned into theabove transformation vectors in sense, or antisense orientation.Alternatively, polynucleotides based on the Arah3 and Arah4 homologousregion may be used for allergen gene control, such as synthesis of 100base pair oligonucleotides within the region of homology and synthesisof at least 100 base pair oligonucleotides at the 5′ end of the cDNA.These oligonucleotides are used in the same way as the PCR products.

Other polynucleotides are also used to down-regulate Arah1. Twodifferent Arah1 clones (Arah1 P41B, and Arah1 P17) were identified inpeanut (Burk et al, 1995). The cDNA sequences of these two clones show96% homology, as highlighted by capital letters in the sequence shown inFIG. 5. A portion of at least two hundred base pairs within the homologyregion is PCR amplified, and then cloned into transformation vectors insense, and antisense orientations. Other polynucleotides that can beused to regulate Arah1 expression include 100 base pair oligonucleotideswithin the region of homology, and 100 or more base pairoligonucleotides at the 5′ end of the cDNA of each clone.

The Ara h5 cDNA sequence does not have any homology with other peanutallergens. FIG. 7. shows the PCR amplified region for the antisense andsense constructs (shown in bold in the sequence) to down-regulate Arah5proteins in peanut plants.

6. Transgenic Peanut Plants

The allergen-free peanut seed according to the present invention may beproduced in essentially any of the various transformation methods knownto those skilled in the art of plant molecular biology. (See, forexample, Wu and Grossman, (Eds.) 1987, Methods of Enzymology, Vol. 153,Academic Press, incorporated herein by reference). As used herein, theterm “transformation” refers to alteration of the genotype of a hostplant by the introduction of non-native or native nucleic acidsequences. Particle bombardment of embryogenic callus, or agrobacteriumtransformation, are the methods of choice for production of transgenicmonocotyledonous plants, but has found widespread application fortransformation of dicotyledonous plants as well. (Vasil, 1994, PlantMol. Biol. 25, 925-937). In many cases transformed plant cells may becultured to regenerate whole plants which can subsequently reproduce togive successive generations of genetically modified plants.

Experiments have shown that foreign genes can be transferred to peanutusing Agrobacterium mediated transformation (Lacorte et al., 1991, PlantCell Reports 10:354-357. Cheng et al., 1991, Proc. Amer. Peanut Res.Educ. Soc., 23:30.) or microprojectile bombardment (Cheng et al., 1991,supra; Ozias-Akins et al., 1993, Plant Science 93:185-194). Themicroprojectile bombardment protocol was reported to produce stablytransformed peanut plants.

To commence a transformation process in accordance with the presentinvention, it is first necessary to construct a suitably modified vectorand properly introduce the vector into the plant cell. The details ofthe construction of the vectors utilized herein are known to thoseskilled in the art of plant genetic engineering.

For example, the allergen-antisense containing constructs utilized inthe present invention can be introduced into plant cells using Tiplasmids, root-inducing (Ri) plasmids, and plant virus vectors. Forreviews of such techniques see, for example, Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, N.Y., SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9, and Florsch et al., Science 227:1229(1985), incorporated herein by reference.

One of skill in the art will be able to select an appropriate vector forintroducing the nucleic acid sequences of the invention in a relativelyintact state. Thus, any vector which will produce a plant carrying theintroduced DNA sequence should be sufficient. Even a naked piece of DNAis expected to be able to confer the properties of this invention,though at low efficiency. The selection of the vector, or whether to usea vector, is typically guided by the method of transformation selected.

For example, a heterologous nucleic acid sequence can be introduced intoa plant cell utilizing Agrobacterium tumefaciens containing the Tiplasmid. When using an A. tumefaciens culture as a transformationvehicle, it is most advantageous to use a non-oncogenic strain of theAgrobacterium as the vector carrier so that normal non-oncogenicdifferentiation of the transformed tissues is possible. It is alsopreferred that the Agrobacterium harbor a binary Ti plasmid system. Sucha binary system comprises 1) a first Ti plasmid having a virulenceregion essential for the introduction of transfer DNA (T-DNA) intoplants, and 2) a chimeric plasmid. The chimeric plasmid contains atleast one border region of the T-DNA region of a wild-type Ti plasmidflanking the nucleic acid to be transferred. Binary Ti plasmid systemshave been shown effective to transform plant cells (De Framond,Biotechnol., 1:262, 1983; Hoekema et al., 1983, Nature 303:179.) Such abinary system is preferred because it does not require integration intoTi plasmid in Agrobacterium.

Methods involving the use of Agrobacterium include, but are not limitedto: 1) co-cultivation of Agrobacterium with cultured isolatedprotoplasts; 2) transformation of plant cells or tissues withAgrobacterium; or 3) transformation of seeds, apices or meristems withAgrobacterium.

In addition, gene transfer can be accomplished by in situ transformationby Agrobacterium, as described by Bechtold et al., 1993, C. R. Acad.Sci. Paris 316:1194. This approach is based on the vacuum infiltrationof a suspension of Agrobacterium cells.

Alternatively, the allergen antisense gene-containing constructdescribed herein can be introduced into a plant cell by contacting theplant cell using mechanical or chemical means. For example, nucleic acidcan be mechanically transferred by direct microinjection into plantcells utilizing micropipettes. Moreover, the nucleic acid may betransferred into plant cells using polyethylene glycol which forms aprecipitation complex with genetic material that is taken up by thecell.

The nucleic acid can also be introduced into plant cells byelectroporation (Fromm et al., Proc. Natl. Acad. Sci., U.S.A. 82:5824(1985), which is incorporated herein by reference). In this technique,plant protoplasts are electroplated in the presence of vectors ornucleic acids containing the relevant nucleic acid sequences. Electricalimpulses of high field strength reversibly permeabilize plant membranesallowing the introduction of nucleic acids. Electroporated plantprotoplasts reform the cell wall, divide and form a plant callus.Selection of the transformed plant cells with the transformed gene canbe accomplished using phenotypic markers as described herein above.

Another method for introducing nucleic acid into a plant cell is highvelocity biolistic penetration by small particles with the nucleic acidto be introduced contained either within the matrix of small beads orparticles, or on the surface thereof (Klein et al., 1987, Nature 327:70.Although, typically only a single introduction of a new nucleic acidsequence is required, this method particularly provides for multipleintroductions.

Cauliflower mosaic virus (CaMV) may also be used as a vector forintroducing heterologous nucleic acid into plant cells (U.S. Pat. No.4,407,956). The CaMV viral DNA genome is inserted into a parentbacterial plasmid creating a recombinant DNA molecule which can bepropagated in bacteria. After cloning, the recombinant plasmid may bere-cloned and further modified by introduction of the desired nucleicacid sequence. The modified viral portion of the recombinant plasmid isthen excised from the parent bacterial plasmid, and used to inoculatethe plant cells or plants. Plasmids pCB13, pBI426, and pBI434 may alsobe used as vectors for introducing heterologous nucleic acids intoplants. Peanut allergen genes are cloned into these vectors in sense orantisense orientation for single transformations or multipletransformations (co-bombardments). (Chen et al., 1998 NatureBiotechnology 16: 1060-1064; Pawloski, Somers et al., 1996 MolBiotechnol 6:17-30) Using Agrobacterium Ti vector-mediated planttransformation methodology, all polynucleotide molecules of thisinvention can be inserted into peanut genomes after the polynucleotidemolecules have been placed between the T-DNA border repeats of suitabledisarmed Ti-plasmid vectors (Deblaere, R. et al., 1987, Methods inEnzymology 153 277-292). This transformation can be carried out in aconventional manner, for example as described in EP 0116718, PCTpublication WO 84/02913 and EPA 87400544.0. The polynucleotide moleculecan also be in non-specific plasmid vectors which can be used for directgene transfer (e.g. de la Pena, A., 1987, Nature, 325:274-276).

As indicated above, the polynucleotide molecule according to the instantinvention preferably encodes antisense RNAs to all peanut allergengenes, including Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7,and any other peanut allergen genes.

Alternatively, the skilled person will appreciate that nucleotidesequences as defined herein may be introduced into a peanut cell genomewhich already comprises one or more antisense, or sense, or acombination of sense and antisense allergen genes. Specifically, thepolynucleotide molecule may contain the sequence encoding one of theantisense RNAs, the sense RNA, and sequential transformation(retransformation) may be used to introduce a second polynucleotidemolecule comprising a different sense or antisense allergen gene. Inthis case, an alternative system to select transformants is needed forthe second round of transformation. For example, two differentselectable marker genes are used in the two consecutive transformationsteps. The first marker is used for selection of transformed cells inthe first transformation, while the second marker is used for selectionof transformants in the second round of transformation. Sequentialtransformation steps using kanamycin and hygromycin have been described,for example by Sandler et al. (1988) and Delauney et al. (1988). Thisretransformation leads to the combined expression of two sense orantisense genes, or some combination thereof, resulting in a transgenicplant that is free of or has reduced or undetectable expression of, morethan one allergen. This transformation step can be repeated until allknown peanut allergen genes have been silenced and the resultedtransgenic peanut plant is completely free of known existing allergens.

Another alternative is to transform a peanut plant using apolynucleotide molecule containing a sequence encoding one antisenseRNA, or one sense RNA, and/or a combination of antisense and sense RNA,and transform another peanut plant using a second polynucleotidemolecule containing a sequence encoding the other antisense RNA, senseRNA, and/or a combination of antisense and sense RNA, in a single plantgenome through crosses of the two independently transformed peanutplants. It is well-known to a skilled person that the plants, prior tocrossing, should be rendered homozygous regarding the transgene throughselfing. The first plant should be a plant transformed with a firstantisense RNA or a sense RNA, and/or a combination of antisense andsense RNA, or an F1 plant derived thereof through selfing. Selectionmethods can be applied to the plants obtained from this cross in orderto select those plants having the two antisense RNAs genes, sense RNAgenes, and/or a combination of antisense and sense RNA genes present intheir genome (e.g. by Southern blotting) and blocking the expression ofone or more allergens (e.g. by separate ELISA detection). A skilledartisan would recognize that this strategy can be repeated, such thatfurther antisense genes, sense genes, and/or a combination of antisenseand sense genes, are introduced sequentially by crossing.

7. Allergen-Free Transgenic Peanut Method to Render the AntisenseTransgene Homologous, and Use of Allergen Free Peanut in TraditionalBreeding Programs

Allergen-free germplasm may be incorporated in traditional breedingprograms for incorporation of this novel trait into desirable peanutgenotypes. Methods for producing novel, allergen-free peanut hybridsusing the transgenic allergen-free peanut plant of the present inventionare known in the art. Each of the following references is incorporatedin its entirety, herein, by reference: Moore, 1989, K. M. et al., J.Heredity 80(3): 252; Norden, A. J., Peanuts, Culture and Uses. Am.Peanut Res. and Educ. Soc., Stillwater, Okla. (C. T. Wilson ed. 1973);Norden, A. J. in Hybridization of Crop Plants (H. H. Hadley ed. 1980);Norden, A. J., et al., Breeding of the cultivated peanut in PeanutScience and Technology, (H. E. Pattee ed. 1992); Norden, A. J. et al.Florida Agr. Res. 3:16-18 (1984).

Initially, a homozygous line containing the antisense allergen gene canbe obtained, following conventional peanut breeding by self-pollinationfor a number of generations. This homozygous line may be introgressedinto diverse peanut backgrounds in the same, or different market classesby breeding methods known in the art, such as successive selection andinbreeding.

The allergen-free peanut germplasm of the present invention can beintrogressed into diverse peanut backgrounds in the same, or differentmarket classes, for example, the runner-type market class (A. hypogaeasubsp. hypogaea var. hypogaea botanical type Virginia) as well as theVirginia (A. hypogaea subsp. hypogaea var. hypogaea botanical typeVirginia), Peruvian (A. hypogaea subsp. hypogaea var. hypogaea botanicaltype Peruvian runner), Valencia (A. hypogaea subsp. fastigata var.fastigata botanical type Valencia) and Spanish (A. hypogaea subsp.fastigata var. vulgaris botanical type Spanish) market classes. Peanutsin the runner-type market class are the most commonly used varieties andare found in diverse products such as peanut butter, salted nuts andconfectionery products. On the other hand, peanut varieties in theVirginia market class are largely used as salted nuts and in-shellmarket. The Valencia is largely used in peanut butter while the Spanishtype is used in certain niche markets where small round peanuts areneeded such as confectionery products and red skin peanuts. Finally, thePeruvian runner market class is grown in certain regions of Mexico.

The allergen-free peanut germplasm of the present invention isintrogressed into different peanut backgrounds by conventional methodswell know to the skilled artisan in the field of peanut breeding. Morespecifically, crosses are made according to methods described by Norden,A. J., Peanuts, Culture and Uses, supra. Am. Peanut Res. and Educ. Soc.,Stillwater, Okla. (C. T. Wilson ed. 1973); Norden, A. J. inHybridization of Crop Plants (H. H. Hadley ed. 1980); Norden, A. J., etal., Breeding of the cultivated peanut in Peanut Science and Technology,(H. E. Pattee ed. 1992); Norden, A. J. et al. Florida Agr. Res. 3:16-18(1984), the entirety of each is incorporated by reference. Introgressionof the allergen-free characteristic is via the traditional plantbreeding cross pollination techniques.

Allergen-free peanut plants may be propagated by planting homozygousseeds and harvesting the crop.

8. Production of Foods Using Allergen-Free Peanut

Allergen free peanuts produced according to the instant invention areprocessed and manufactured into food products using methods well knownto a skilled artisan. Allergen-free peanut products are produced usingthe same standard food processing methods, processing equipment andsanitation practices, as those used in the production of theirnon-allergen-free counterparts. A skilled artisan would recognize thatmanaging the risk of cross contamination in a food plant producingallergen-free peanut products is critical. If possible the system shouldbe dedicated to producing only allergen-free foods (Beckman and Coult,1999, Food Testing & Analysis 5 (3): 15-17).

9. Protein Expression

Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Polypeptides of the invention can also include aninitial modified methionine residue, in some cases as a result ofhost-mediated processes.

Purified proteins of the present invention may be used in the treatmentof individuals allergic to peanut allergens, for example, viapercutaneous specific hyposensitization therapy (see, e.g. Kaneko etal., U.S. Pat. No. 5,951,984) or via oral hyposensitization therapy(Wells et al., 1991, J. Infect. Dis., 8:66; Trentham et al., 1993,Science, 261:1727; Weiner, et al., 1993, Science, 259:1321).

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian, or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or inducible), followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a strong promoter to direct transcription, aribosome binding site for translational initiation, and atranscription/translation terminator. One of skill would recognize thatmodifications can be made to a protein of the present invention withoutdiminishing its biological activity. Some modifications may be made tofacilitate the cloning, expression, or incorporation of the targetingmolecule into a fusion protein. Such modifications are well known tothose of skill in the art and include, for example, a methionine addedat the amino terminus to provide an initiation site, or additional aminoacids (e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambdaderived P L promoter and N-gene ribosome binding site (Shimatake et al.,Nature 292:128 (1981)). The inclusion of selection markers in DNAvectors transfected in E. coli is also useful. Examples of such markersinclude genes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., 1983, Gene22: 229-235; Mosbach, et al., 1983, Nature 302: 543-545).

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a of the present invention can beexpressed in these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous proteins in yeast is well known. Sherman, F.,et al., 1982, Methods in Yeast Genetics, Cold Spring Harbor Laboratoryis a well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeast for productionof eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques or radioimmunoassayof other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cells. Mammalian cell systems often will be in the form ofmonolayers of cells although mammalian cell suspensions may also beused. A number of suitable host cell lines capable of expressing intactproteins have been developed in the art, and include the HEK293, BHK21,and CHO cell lines. Expression vectors for these cells can includeexpression control sequences, such as an origin of replication, apromoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., 1986,Immunol. Rev. 89: 49), and necessary processing information sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., an SV40 large T Ag poly A addition site), and transcriptionalterminator sequences. Other animal cells useful for production ofproteins of the present invention are available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(7th edition, 1992).

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See Schneider,1987, J. Embryol. Exp. Morphol. 27: 353-365.

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,1983, J. Virol. 45: 773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo, M.,Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA CloningVol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington,Va. pp. 213-238 (1985).

A peanut allergen protein can be recovered and purified from recombinantcell cultures by well known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography (“HPLC”) is employed for purification.

The monitoring of the purification process can be accomplished byWestern blot techniques, radioimmunoassay, or other standard immunoassaytechniques. These methods are described in many standard laboratorymanuals, such as Sambrook, supra, Chapters 17.37-17.42; Ausubel, supra,Chapters 10, 12, 13, 16, 18 and 20.

10. Antibodies of the Invention

Antibodies raised against the proteins and protein fragments of theinvention also are contemplated by the invention. In particular, theinvention contemplates antibodies raised against the peanut allergenprotein Ara h2, and variants thereof. Described below are antibodyproducts and methods for producing antibodies capable of specificallyrecognizing one or more epitopes of the presently described proteins andtheir derivatives. Antibodies include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies including single chain Fv (scFv)fragments, Fab fragments, F(ab′)₂ fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, epitope-bindingfragments, and humanized forms of any of the above.

As known to one in the art, these antibodies may be used, for example,in the detection of a target protein in a food sample. It is importantfor peanut-sensitive individuals to have a means of recognizing andavoiding peanut-containing products. Unfortunately, peanut allergenshave been identified in non-peanut foodstuffs manufactured on commonprocessing equipment that were inadequately cleaned. In general, Keatinget al., 1990, J. Allerg. Clin. Immunol., 86: 41-4, which is hereinincorporated by reference, describes the use of a radioimmunoassay todetect peanut allergens in food processing materials and finished foods.In one embodiment, the allergen is covalently coupled to a solid phase.The allergen is reacted with a patient serum sample containing bothallergen specific and non-specific IgE. The allergen reacts with thespecific IgE in the patient sample. After washing away non-specific IgE,radioactively labeled antibodies against IgE are added forming acomplex. Then unbound radioactively labeled anti-IgE is washed away. andthe radioactivity of the bound complex is measured, for example, in agamma counter. The more bound radioactivity found, the more specific IgEpresent in the sample. To classify the test results, patient counts arecompared directly with counts of reference sera run in parallel. Inanother embodiment, the “two-site monoclonal antibody enzyme-linkedimmunosorbent assay” described in Burks et al. (U.S. Pat. No. 5,558,869)is used for the detection of the allergen.

The antibody of the present invention may also be utilized as part oftreatment methods. For example, Saint-Remy et al., in U.S. Pat. No.5,026,545 describes a method for treating allergic reaction viaadministering to a patient a mixture of allergen-antibody complex.

In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, A. M., 1984, MonoclonalAntibody Technology: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands; St.Groth et al., 1980, J. Immunol. Methods 35:1-21; Kohler and Milstein,1975, Nature 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72; Cole etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

i) Polyclonal Antibodies

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as an inventive protein or an antigenic derivative thereof.Polyclonal antiserum, containing antibodies to heterogenous epitopes ofa single protein, can be prepared by immunizing suitable animals withthe expressed protein described above, which can be unmodified ormodified, as known in the art, to enhance immunogenicity. Immunizationmethods include subcutaneous or intraperitoneal injection of thepolypeptide.

Effective polyclonal antibody production is affected by many factorsrelated both to the antigen and to the host species. For example, smallmolecules tend to be less immunogenic than other and may require the useof carriers and/or adjuvant. In addition, host animal response may varywith site of inoculation. Both inadequate or excessive doses of antigenmay result in low titer antisera. In general, however, small doses (highng to low μg levels) of antigen administered at multiple intradermalsites appears to be most reliable. Host animals may include but are notlimited to rabbits, mice, and rats, to name but a few. An effectiveimmunization protocol for rabbits can be found in Vaitukaitis, J. etal., 1971, J. Clin. Endocrinol. Metab. 33:988-991.

The protein immunogen may be modified or administered in an adjuvant inorder to increase the protein's antigenicity. Methods of increasing theantigenicity of a protein are well known in the art and include, but arenot limited to coupling the antigen with a heterologous protein (such asglobulin β-galactosidase) or through the inclusion of an adjuvant duringimmunization. Adjuvants include Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

Booster injections can be given at regular intervals, with at least oneusually being required for optimal antibody production. The antiserummay be harvested when the antibody titer begins to fall. Titer may bedetermined semi-quantitatively, for example, by double immunodiffusionin agar against known concentrations of the antigen. See, for example,Ouchterlony et al., 1973, Chap. 19 in: Handbook of ExperimentalImmunology, Wier, ed, Blackwell. Plateau concentration of antibody isusually in the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Theantiserum may be purified by affinity chromatography using theimmobilized immunogen carried on a solid support. Such methods ofaffinity chromatography are well known in the art.

Affinity of the antisera for the antigen may be determined by preparingcompetitive binding curves, as described, for example, by Fisher, 1980,Chap. 42 in: Manual of Clinical Immunology, second edition, Rose andFriedman, eds., Amer. Soc. For Microbiology, Washington, D.C.

ii) Monoclonal Antibodies

Monoclonal antibodies (MAbs), are homogeneous populations of antibodiesto a particular antigen. They may be obtained by any technique thatprovides for the production of antibody molecules by continuous celllines in culture or in vivo. MAbs may be produced by making hybridomas,which are immortalized cells capable of secreting a specific monoclonalantibody.

Monoclonal antibodies to any of the proteins, peptides and epitopesthereof described herein can be prepared from murine hybridomasaccording to the classical method of Kohler, G. and Milstein, C., 1975,Nature 256:495-497; and U.S. Pat. No. 4,376,110 or modifications of themethods thereof, such as the human B-cell hybridoma technique (Kosbor etal., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad.Sci. USA 80: 2026-2030), and the EBV-hybridoma technique (Cole et al.,1985, Monoclonal antibodies and cancer therapy, Alan R. Liss, Inc., pp.77-96).

In one method a mouse is repetitively inoculated with a few microgramsof the selected protein over a period of a few weeks. The mouse is thensacrificed, and the antibody producing cells of the spleen are isolated.

The spleen cells are fused, typically using polyethylene glycol, withmouse myeloma cells, such as SP2/0-Ag14 myeloma cells. The excess,unfused cells are destroyed by growth of the system on selective mediacomprising aminopterin (HAT media). The successfully fused cells arediluted, and aliquots are plated to microliter plates where growth iscontinued. Antibody-producing clones (hybridomas) are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures. These include ELISA, as originally described byEngvall, 1980, Meth. Enzymol. 70:419, western blot analysis,radioimmunoassay (Lutz et al., 1988, Exp. Cell Res. 175:109-124) andmodified methods thereof.

Selected positive clones can be expanded and their monoclonal antibodyproduct harvested for use. Detailed procedures for monoclonal antibodyproduction are described in Davis, L. et al. 1989, Basic methods inmolecular biology, Elsevier, N.Y. Section 21-2. The hybridoma clones maybe cultivated in vitro or in vivo, for instance as ascites. Productionof high titers of mAbs in vivo makes this the presently preferred methodof production. Alternatively, hybridoma culture in hollow fiberbioreactors provides a continuous high yield source of monoclonalantibodies.

The antibody class and subclass may be determined using procedures knownin the art (Campbell, 1984, Monoclonal Antibody Technology: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands). MAbs may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. Methods of purifying monoclonal antibodies are well known inthe art.

EXAMPLES

The following examples are given to illustrate the present invention. Itshould be understood that the invention is not to be limited to thespecific conditions or details described in these examples. Throughoutthe specification, any and all references to publicly availabledocuments are specifically incorporated by reference

Example 1 Isolation and Characterization of the Genomic Clones Encodingthe Peanut Allergen Genes

a) Library screening

To identify the genomic clone of the gene coding for the peanut allergenAra hII, a peanut genomic library constructed in a Lambda Fix II vector(Stratagene Inc, La Jolla, Calif.) was screened with an 80 base pairoligonucleotide probe. The probe sequence(5′ctagtagccctcgcccttttcctcctcgctgcccacgcatctgcgaggcagcagtgggaactccaaggagacagaagatg-3′)(SEQ ID NO: 7) corresponds to nucleotide eleven to ninety-one of apublished Ara h2 cDNA sequence (GeneBank accession L77197).

Twenty picomoles of the probe was end-labeled with radioactive adenosine5′-triphosphate, tetra (triethylammonium), salt [gamma 32P] (32P) asdescribed by Ausubel et al. (Ausubel F, Brent R, Kingston R E, Moore DD, Seidman J G, Smith J A, Struhl K. Short Protocols in MolecularBiology. 3rd ed.: John Wiley & Sons, Inc.; 1995) Fresh Echerichia Coli(E. Coli) VCS 257 (300 μL of 1×1010 cells/mL) were infected with 10 μLof the genomic library (1×103 pfu) for 30 minutes at 37° C. in a waterbath. Then, 7 mL of top agarose (0.7%) at 47° C. were added, mixed andspread onto a pre-warmed (37° C.) 150 mm 2×LB agar plate. (Sambrook J,Fritsch E F, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed.New York: Cold Spring Harbor Laboratory Press; 1989) The plaques becamevisible after an overnight incubation at 37° C.

After plaque formation, the culture dishes were stored for 4 hours at 4°C., blotted on a piece of nylon membrane, denatured (NaOH, 0.5N) andneutralized (Tris-HCl, 1M) according to manufacturer's instructions (NENLife Science Products, Inc., Boston, Mass.) and the DNA was crosslinkedat 12,000 μjoules of UV energy for 45 seconds (UV Stratalinker 1800,Stratagene). Low stringency prehybridization (at 42° C. for 3 hours) andhybridization (at 42° C. overnight) were performed in the same solutioncontaining 50% (v/v) formamide+10% (w/v) SDS+20% (w/v) dextransulfate+1×Denhardt's solution+10 μg/mL salmon sperm DNA. Duringhybridization the labeled probe was added to the buffer. Membranes werewashed with 2×SSC followed by 2×SSC+0.1% SDS for 15 minutes at roomtemperature, air dried, exposed to Kodak XAR-5-X ray film and developedafter seven days at −80° C. Positive clones were matched with plaques onthe Petri dishes, lifted and stored at 4° C. in 1 mL SM media containinga few drops of chloroform to prevent bacterial contamination. (Sambrooket al., supra) To confirm true positive clones, a second screening isperformed as described above.

b) Purification of Putative Positive Clones

Selected putative positive clones were amplified as described bySambrook et al. (Sambrook et al., supra) Lysate stocks of recombinantbacteriophage were prepared by infection of E. coli VCS 257 with eachputative positive clone. The culture was grown for 6-8 hours at 37° C.and 300 rpm. Purification of lambda DNA was done using a Lambda kit(Qiagen Inc., Valencia Calif.) and the DNA was quantified using afluorometer. (Hoefer Scientific Instruments. TKO 100DNA Mini FluorometerInstruction Manual 1991)

c) Dot Blot Analysis

Positive clones were confirmed by dot blot analysis using a Bio-Dot SFMicrofiltration apparatus (Bio-Rad Laboratories, Inc., Hercules, Calif.)and the Southern hybridization protocol. (Southern, E M., J Mol Biol1975; 98(3): 503-517) One microgram of each purified DNA was blotted andtransferred by capillary action to a nylon membrane. DNA was crosslinkedto the membranes at 12,000 μjoules of UV energy for 45 seconds. Themembrane was prehybridized at 50° C. for a least 4 hours in6×SSPE+5×Denhardt's solution+0.05% (w/v) NaPyrPO4+0.5% (w/v) SDS+100μg/mL salmon sperm DNA and hybridized at 50° C. overnight in6×SSPE+1×Denhardt's solution+0.05% NaPyrPO4+0.5% SDS with the same 32Pend-labeled probe used to screen the library. Stringent washes wereperformed at 50° C. for 15 minutes each in 6×SSPE+0.1% (w/v) SDS and2×SSPE+0.1% (w/v) SDS. After air drying, the membrane was exposed toKodak X-Omar AR film at −80° C. for two days and autoradiographed.

d) Subcloning

The selected positive lambda clone for Ara h2 was subcloned into apBluescript II SK(+/−) phagemid vector (Stratagene, La Jolla, Calif.) tofacilitate sequencing.

e) Subcloning of a 12 kb Fragment into a Phagemid Vector

The selected positive lambda clone was approximately 50 Kb with aninsert fragment of about 16 Kb. The clone was digested with BamH I torelease the insert and electrophoresed on a 0.7% agarose gel. Fivefragments ranging in size from 5.5, 6.5, 9, 12 and 16 Kb were obtained.After Southern hybridization, only the 12 kb fragment hybridized to the32P-labeled 80-mer probe. The 12 Kb fragment was then gel purified andsubcloned into a pBluescript II SK+ plasmid vector (FIG. 1). Sequenceanalysis revealed that the selected 12 kb DNA fragment is truncated at aBamH I restriction site located about 212 nucleotides within the gene.

f) Subcloning of a 6.5 kb Fragment into a Phagemid Vector

A 62 base pair probe(5′-gtgcatgtgcgaggcattgcaacagatcatggagaaccagagcgataggttgcaggggaggc-3′)(SEQ ID NO: 8) was designed from cDNA sequence downstream from the BamHI site to capture the remaining DNA fragment of the Ara hII gene. Of thefive fragments obtained after digestion of the 50 kb lambda clone withBamH I, only the 6.5 kb fragment hybridized to this probe. This fragmentwas subcloned into pBluescript II SK+ plasmid vector and sequenced (FIG.1).

g) Restriction Enzyme Digestion

For the BamH I digestion, the clone was electrophoresed on a 0.7%agarose gel. Five fragments ranging in size from 5.5, 6.5, 9, 12 and 16Kb were obtained. After Southern hybridization, only the 12 kb fragmenthybridized to the 32P-labeled 80-mer probe, and was then gel purifiedand subcloned into a pBluescript II SK+ plasmid vector (FIG. 1).Sequence analysis revealed that the selected 12 kb DNA fragment istruncated at a BamH I restriction site located about 212 nucleotideswithin the gene.

Restriction enzyme digestion with BamH I was performed at 37° C.Fragments were separated by electrophoresis on a 0.7% agarose gel, andfive fragments, 5.5, 6.5, 9, 12 and 16 kb, were obtained. Each fragmentwas cut from the agarose gel and filtered through a MilliporeUltrafree®-DA filter (Millipore Corp., Bedford, Mass.) and precipitatedin 100% ethanol. The digested pBluescript II vector was dephosphorylatedwith calf intestinal alkaline phosphatase prior to ligation with the DNAfragments, purified with an equal volume of phenol-chloroform, andprecipitated in ethanol and resuspended in one volume of TE buffer (5 mMTris (pH 7.5, 0.1 mM EDTA) to a final concentration of approximately 0.1μg/μL.

h) Ligation

A 2:1 and 3:1 ratio of insert to vector DNA was selected. The ligationreaction was performed at 4° C. overnight then at room temperature forthree hours. About 20 μL of ultra competent bacteria cells GENEHOGS™Research Genetics (E. coli DH10B) were mixed with 1 μL of ligationmixture, electroporated and resuspended in 1 mL of 37° C. sterile SOCmedium as described in the GENEHOGS™ protocol (Research Genetics,Huntsville, Ala.). Electroporation was performed using a Bio-Rad GenePulser electroporator (Bio-Rad Laboratories, Richmond, Calif.) with thefollowing settings for a 1 mm gap electroporation cuvette(BTX™Genetronics, Inc, San Diego, Calif.): the field strength at 17kV/cm, the resistor at 200Ω and the capacitor at 25 μF. Positivecolonies were selected by blue-white color selection. (StratageneIncorporation. Instruction manual: pBluescript®IIExo/Mug DNA SequencingSystem. 1999) From each plate, white positive colonies containing aplasmid with an insert were picked and placed onto 6 mL of LB mediasupplemented with ampicillin (100 μg/mL) and incubated at 37° C. for 16hours at 300 rpm. Plasmid DNA was purified using Qiagen PlasmidPurification kit, digested with BamH I and separated on 0.7% agarose gelto confirm the presence of a plasmid containing an Ara h2 insert.

i) Southern Hybridization

Digested DNA fragments were transferred onto a nylon membrane using analkaline transfer protocol according to manufacturer instructions (Pall,NEN™ Life Science Products, Inc., Boston, Mass.). The DNA wascrosslinked on the membrane as previously described and pre-hybridizedat 65° C. for 3 hours in HyperHyb buffer (Research Genetics, Inc.,Huntsville, Ala.). The probe was end labeled with 32P as described inthe Fermentas kit (Fermentas Inc., Hanover, Md.), added to thehybridization solution and incubated at 65° C. for 3 hours in HyperHybbuffer. The membrane was washed three times at 65° C. for 15 minuteseach in 0.1×SSC+0.1% SDS, rinsed once at room temperature in 1×SSC andexposed to x-ray film (Kodak, Biomax™ MS) at −80° C. for three hours andautoradiographed.

j) Sequencing

Purified positive p-Bluescript DNA (0.2 μg/μL) were sequenced with ABIPRISM™ Dye Terminator Cycle Sequencing Ready Reaction kit usingAmpliTaq® DNA Polymerase, FS at Research Genetics, Inc. and theUniversity of Alabama in Birmingham (UAB) using T3 and T7 sequencingprimers.

k) Sequence Analysis

Approximately 1.2 kb of the peanut genomic DNA inner than beencompletely sequenced for both the sense and antisense strands, as can beseen in FIG. 2. It has been determined that Ara h2 is a gene family andcontains iso-forms of the gene. Southern Blot analysis and thedifference between the originally characterized cDNA clone and thecharacterization of the genomic clone of the present invention, isconsistent with the existence of multiple genes.

Analysis of the sequence reveals a full length Ara h2 gene. Sequenceanalysis, comparison and homology searches are performed using the BLAST(Altschul S F, Madden T L, Schäffer A A, Zhang J, Zhang Z, Miller W,Lipman D J., Nucl Acids Res 1997; 25:3389-3402), and BLAST 2 sequencestools. (Tatusova T A, Madden T L., FEMS Microbiol Lett 1999;174(2):247-250) Determination of leader sequence is done as described byGrierson and Covey. (Grierson D, Covey S N. Plant Molecular Biology. 2nded. New York (N.Y.): Chapman and Hall Publishers; 1988)

As evident from inspection of the sequence shown in FIG. 2, the openreading frame of the gene starts with a initiation codon (ATG) atposition 1 and ends with a termination codon (TGA) at position 622. Thepredicted encoded protein is 207 amino acids long and includes aputative transit peptide of 21 residues.

One putative polyadenylation signal AATAAA is identified at position951. Six additional putative stop codons are observed downstream of thefirst termination codon at positions 628 (TGA), 769 (TAA) 901 (TAA), 946(TGA), 967 (TGA) and 982 (TGA). In the promoter region, 5′ upstream ofthe start codon, a putative TATA box, TATTATTA is present at position−72. Comparison of the published cDNA and genomic sequences revealed theabsence of an intron.

The location of the initiation codon ATG of Ara h2 is revealed for thefirst time. Until now only partial cDNA sequences have been published.(Stanley J S, King N, Burks A W, Huang S K, Sampson H, Cockrell G, HelmR M, West M, Bannon G A. Arch Biochem Biophys 1997; 342(2):244-253) Theopen reading frame of the genomic clone of Ara h2 is 621 nucleotideslong while its cDNA (GeneBank accession L77197) is 492 nucleotides long.A comparison of the 2 sequences reveals that the cDNA sequence is 8nucleotides short at the 5′ region and does not include a start codon.In addition, the two sequences have complete identity from nucleotide 9to 470 of the genomic clone. However, from nucleotide 471 they divergewith no homology downstream from this region at the nucleotide as wellas the amino acid levels.

The termination codon is TGA at position 622. Not only is thetermination codon usage different between the genomic (TGA) and the cDNA(TAA) clone but the later also ends 152 by or 51 amino acids earlierthan the genomic clone. Six additional stop codons are present in the 3′untranslated region at positions 628 (TGA), 769 (TAA), 901 (TAA), 946(TGA), 967 (TGA) and 982 (TGA). It is known that some genes have severaltermination codons (Grierson & Covey, supra), however it is unclearwhich one is preferentially used. A gene usually undergoes posttranscriptional and post translational modifications, which couldexplain some of the differences between the genomic and cDNA sequences.

A putative polyadenylation signal AATAAA is located at position 951 inthe 3′ untranslated region of the gene. This signal is identical to theconsensus sequence for plants. Polyadenylation signals play key role inthe stability and translation of the genetic message and direct thetermination of transcription by RNA polymerase II (a functionalpolyadenylation signal and a downstream transcription ‘pause’ elementare required for efficient pol II transcription termination in fissionyeast. See Birse C, Proudfoot N.,genome-www.stanford.edu/Saccharomyces/yeast96/f2021.html; Poly(A) signalcontrols both transcriptional termination and initiation between thetandem GAL10 and GAL7 genes of Saccharomyces cerevisiae. Greger I H,Proudfoot N J., EMBO J. 1998; 17(16):4771-4779).

FIG. 2 shows the deduced polypeptide encoded by the open reading framewhich has 207 amino acids residues and includes a putative signalpeptide of 21 amino acid residues (Nielsen H, Engelbrecht J, Brunak S,von Heijne G. Protein Engineering 1997, 10:1-6.). A signal peptide playsa role in the translocation of a protein from the cytosol to the targetorganelle within the cell. (Alberts B, Bray D, Lewis J, Raff M, RobertsK, Watson J D. Molecular Biology of the Cell. 3rd ed. New York (N.Y.):Garland Publishing, Inc; 1994) It is typically composed of hydrophobicamino acids such as tryptophan, phenylalanine, valine, leucine andisoleucine that have affinity for membranes of organelles. Ibid.

In the proximal region of the promoter, a putative TATA box TATTATTA ispresent at position −72 with respect to the initiation codon. Theconsensus signal for plant TATA boxes is TATAT/AA1-328. This is the mostconserved sequence for RNA polymerase II-mediated transcription and isimportant for positioning the start of transcription. (Alberts et al.supra; Ellison K, Messing J., Biotechnology 1983; 12:115-139).

The 3′ end of the Ara h2 gene (as shown in FIG. 2, downstream of thestop codon of the gene itself) can be fused, or operably linked, to aheterologous gene for expression of that gene.

Example 2 Construction Strategy of Peanut Allergen Gene Plasmids

Peanut allergen gene plasmids were constructed using expressioncassettes containing antisense, and/or sense orientation of allergengenes linked to 35S/Ara h2 promoter and nos terminator, as shown in FIG.8. Five types of constructs were used for the transformation of peanuttissue. The plasmid constructs pBI426, modified pBI426, and pCB13, wereused in biolistic transformation. pBI434modified pBI434 were used inAgrobacterium-mediated transformation The Ara h2 promoter is shown inFIG. 9.

PCB13 is used in co-bombardments with modified pBI426 which containspeanut allergen fragments (Ara h transgenes), to select transgenicplants. pCB13 contains the 35S promoter, hygromycin gene, and the nosterminator. This cassette is cloned into pUC19.

The plasmid pBI426 (Dalta et al., 1991 Gene 101: 239-246) contains afusion gene (GUS fused to nptII) cloned between XbaI and SacI, anddriven by the 35S promoter. Its also contains a nontranslatable leadersequence of 50 base pairs from Alfalfa Mosaic Virus. The entireexpression cassette is cloned into pUC18.

In the modified pBI426 plasmid, for peanut transformation the gus-nptIIfusion gene is replaced with DNA sequences from peanut allergens eitheras PCR products, or synthetic oligonucleotides. Ara h transgenes areclones as XbaI/SacI fragments in sense and antisense orientations. Forcomparative studies, 35S promoter is replaced with Arah2 promoter (shownin FIG. 9).

For Agrobacterium-mediated transformation of peanut, plasmid constructspBI434 and modified pBI434 were used.

pBI434 is a binary vector derived from pBIN19. The T-DNA between theright border (RB) and the left border (LB) contains the 35S promoter,the 50 base pairs leader sequence from alfalfa mosaic virus, the fusiongene gus-nptII, and the nos terminator.

In the modified pBI434, the GUS gene is replaced by allergen DNAsequences (Ara h transgene) as XbaI/PstI fragments in sense andantisense orientations. NptII is the selection marker, and transgenictissues are selected with kanamycin or paromomycin.

Example 3 Methodology Used in Peanut Tissue Culture, Transformation andRegeneration

Peanut Varieties

The most widely cultivated peanut cultivars in the USA, ‘Florunner’,‘New Mexico Valencia’, ‘Georgia Green’, and ‘Georgia Red’ can be used inthe present method, although a person skilled in the art will realizethat the method is applicable to other peanut varieties.

Genetic Constructs

Efficient strategies to down-regulate genes in transgenic plants utilizeantisense RNA, co-suppression, or double-stranded RNA. All three methodsare being used to down-regulate peanut allergens. Transformation vectorsused are pUC18 for biolistic transformation, and modified versions ofpBI434 (Dalta et al, 1991), a binary vector for transformation usingAgrobacterium tumefaciens. Transformation vectors carry the transgenes,flanked by a (the Arah2 promoter, shown in FIG. 9, or the 35S promoter)and the nopaline synthase terminator. Transgenes are portions of theopen reading frame (ORF) of peanut allergens Arah1, Arah2, Arah3, Arah4,Arah5, Arah6, Arah7 genes, and any other peanut allergen genes. Previousstudies show homologies between nucleotide sequences of the genomic DNAof Arah2, and Arah6 and Arah7 cDNAs (Viquez et al, 2000, see FIG. 3).Also, comparison between Arah3 and Arah4 cDNA sequences obtained fromthe gene bank, shows 95% homology. Different types of transformationcassettes are made to down-regulate peanut allergens.

Transformation cassettes type 1 are used to down-regulate Arah2, Arah6and Arah7 in transgenic peanut. The homology region between nucleotidesequences of the above three allergen genes (FIG. 3), is PCR amplifiedfrom the genomic DNA of Arah2. The PCR product is then cloned into thetransformation vectors (pBI426 or pBI434) in the antisense orientationfor the antisense RNA strategy, or in sense orientation for theco-suppression strategy. The PCR product is also operably linked inframe as an antisense and sense fragment for the dsRNA strategy. Also,the antisense construct and sense construct are used inco-transformation for the dsRNA strategy.

Another alternative is to synthesize at least 100 base pairsoligonucleotides corresponding to the homology region, to be used as forthe PCR products.

FIG. 3 shows the nucleotide sequences of the coding region of Arah2genomic DNA (Viquez et al, 2000). The homology region between Arah2,Arah6 and Arah7 is shown in capital letters. This region is amplified byPCR and cloned into the transformation vectors (pUC18, or pBI434).Amplified region to be used for down-regulation of Arah2, Arah6, Ara h7allergens is shown in capital letters. Alternative methods can be usedsuch as 1) to synthesize at least 100 base pairs oligonucleotides withinthe region of homology, 2) to synthesize at least 100 base pairsoligonucleotides at the 5′ end of the cDNA of each allergen gene. Theseoligonucleotides are used in the same way as for the PCR products.

Transformation Cassettes Type 2

Transformation cassettes type 2 are used to down-regulate Ara h3, andAra h4 allergens in peanut. The cDNAs of these two allergens have 95%homology (FIG. 4). A portion of two hundred base pairs within thehomology region is PCR amplified, and then cloned into the abovetransformation vectors in sense, and/or antisense orientation.

FIG. 4 shows the cDNA sequence of Arah h3 (Kleber-Janke, T, 1999). Theamplified region to be used for down-regulation of both Ara h3 and Arah4 allergens is shown in capital letters. Alternative methods that maybe used are 1) to synthesize at least 100 base pairs oligonucleotideswithin the region of homology, 2) to synthesize at least 100 base pairsoligonucleotides at the 5′ end of the cDNA of each allergen gene. Theseoligonucleotides are used in the same way as for the PCR products.

Transformation Vectors Type 3

Transformation vectors type 3 are used to down-regulate the two clonesof Ara h1 (Ara h 1 P41B, and Ara h 1 P17) in peanut. The cDNA sequenceof these two clones show 96% homology. A portion of at least two hundredbase pairs within the homology region is PCR amplified, and then clonedinto the above transformation vectors in sense, and/or antisenseorientation. FIG. 4 shows the nucleotide sequence of Ara h3 41B (Burk etal, 1995). The amplified region to be used for down-regulation of thetwo clones of Ara h1 allergens is shown in capital letters. Alternativemethods that may be used are 1) to synthesize at least 100 base pairsoligonucleotides within the region of homology, 2) to synthesize atleast 100 base pairs oligonucleotides at the 5′ end of the cDNA of eachallergen gene. These oligonucleotides are used in the same way as forthe PCR products.

FIG. 5. shows the sequence of Ara h5 cDNA. This sequence does not haveany homology with other allergens. The PCR amplified region forantisense and/or sense constructs are shown in bold to down-regulate Arah1 proteins in peanut plants.

Promoters

Promoters are the key elements for gene expression. Peanut allergens areseed storage proteins. Therefore, to target the synthesis of peanutallergens, a seed specific promoter is essential. The inventive approachhas developed its own promoter, the Arah 2 promoter, shown in FIG. 9(see also Visqez et al, 2000; which is used to drive RNA transcripts oftransgenes cloned into pBI426 and pBI434. In addition, for comparativestudies, the Arah 2 promoter shown in FIG. 9 is replaced by the 35Spromoter from Cauliflower Mosaic Virus, to compare the degree ofdownregulation. Whichever of the two types of promoter is used, thepromoter is inserted into an expression cassette between HindIII andBglII sites, upstream of and the Ara h transgene PCR amplificationproduct which is inserted between a nontranslatable leader sequence of50 basepairs from Alfala Mosaic Virus, and a nos terminator.

Selection Marker

Plasmid pCB13 lox (see FIG. 8) is provided by Ozias-Akin (University ofGeorgia). This plasmid contains the hygromycin gene, driven by the 35Spromoter. This plasmid is used to select transgenic peanut plants. Inthe case of transformation using Agrobacterium tumefaciens, plasmidpBI434 (Dalta et al., 1991; see FIG. 8) contains the neomycinphosphotransferase II gene for selection with paromomycin or kanamycin.

Reporter Gene

In some experiments, plasmid pBI426 carrying the GUS gene, driven by the35S promoter, is used in co-bombardment with allergen construct, tomonitor transformation in peanut.

Embryogenic cells of peanut variety Georgia green are co-bombarded withpBI426 (containing the gus gene), and pD2, a modified version of pBI426plasmid which contains the sense construct of Ara h2, replacing thefusion gus-nptII gene. Transient GUS assays show transformation eventsas dark blue spots. Cultures are in selection medium. Embryos wereexcised from seeds of peanut cultivars “Florunner” and “Georgia green”.

All three types of transformation vectors described above are used ineach of the strategies of the present invention for antisensetransformation (using antisense constructs), co-suppression (using senseconstructs), and the combining of antisense and sense constructs togenerate in one transformation vector double stranded RNA fusiontranscripts under the control of a single promoter (using either the Arah2 promoter, or the 35S promoter), Constructs for fusion transcripts areused for both biolistic and agrobacterium-mediated transformation.

Peanut Regeneration from Somatic Embryos

Embryos are excised from seeds of peanut, and are sterilized for 30 minin 20% chlorox (v/v) on a shaker at 130 rpm. Seeds are rinsed four timesin sterile distilled water, and embryo axes are then separated fromcotyledons. Embryo axes are plated on MS medium (MS salts and nutrients(Sigma chemical co, St. Louis) supplemented with picloram (3 mg/l),glutamine (1 mg/l), sucrose (20 g/l). pH5.8, before autoclaving. Embryosare cultured, and maintained in dark. After about 3 weeks, proembrymasses are formed on explants. They are subcultured every three weeks onthe same medium.

Transformation

Embryo cultures are bombarded using a PDS1000/helium driven apparentus(Bio-rad Laboratories, Herculus, Calif.). Five μg is of DNA of eachconstruct (single transformations), or five μg of total DNA(co-transformations) are used for biolistic transformation. Goldparticles mixed with the transformation plasmids are accelerated using1100 PSI pressure under a vacuum of 71 cm mercury. One bombardment isconducted for each transformation experiment. After bombardment, embryosare kept on the same medium for 3 days in dark. They are thentransferred in liquid medium supplemented with 20 mg/l hygromycin forselection. The liquid medium is refreshed every two weeks. Cultures aremaintained in dark with shaking at 130 rpm.

Regeneration

Transgenic structures are multiplied on MS+10 mg/l hygromycin+1 mg/lglutamin and maintained in dark for 2-3 weeks. Cultures are thentransferred in light on MS+3 mg/l BAP+1 mg/l GA3 till embryos geminate.Germinated embryos are then transferred on rooting medium (MS+0.2 m/1NAA). Hygromycin is also added to confirm transgenics, becausenon-transgenic plants will not grow.

Peanut Regeneration from Epicotyls

Sterile embryo axes are cultured on MSTDZ medium (MS medium supplementedwith myo-inositol (100 mg/l), sucrose (30 g/l), and thidiazuron (2.2 mlof a 1 mg/ml stock). Culture media are adjusted to pH5.8, beforeautoclaving. Epicotyls are excised from 6 day old germinated embryos byan oblique cut below the cotyledon region, followed by a blunt cut abovethe root axis.

Transformation

Epicotyl sections are washed in ½ MSI solution (MS salts and nutrients(Sigma chemical co, St. Louis) (2.15 g/l and myo-inositol 1 g/l, pH5.8sequentially for three times at 20 min each. Explants are then immersedin a solution of Agrobacterium diluted with MSI at OD600=0.5−1.Agro/explant mixture is swirled for ten min. Explants are then blottedon sterile towel paper, and then transferred on MSO (MS mediumsupplemented with myo-inositol (100 mg/l), sucrose (30 g/l) for fivedays co-culture. After the co-culture period, epicotyl explants aretransferred on MSTDZ medium supplemented with 400 mg/l carbenicillin,and 200 mg/l kanamycin (selection) until shoot formation. Putativetransgenic shoots are excised for transfer on rooting medium MSTDZ+50mg/l kanamycin.

Example 4 Regeneration of Transformed Peanut and Verification ofAntisense Expression and Level of Down Regulation of Peanut Allergens

Transformed epicotyl sections are transferred to MSTDZ mediasupplemented with 200 mg of kanamycin per liter for selection. Allplasmid vectors utilized contain kanamycin resistance genes. Epicotylexplants remain on this media for 2-3 weeks under 16 h/8 h light/darkand 26° C. incubation. Plasmid pBI434 contains the kanamycin resistancegene.

Regenerated shoots are excised and placed on MSO supplemented with 200mg kanamycin per liter for selection. This step is for thedetoxification of thidiazuron since this chemical prohibits rootformation. Regenerated plants are grown on this media for 2-4 weeksunder 16 h/8 h light/dark and 26° C. incubation.

Putative transgenic shoots are moved to rooting media (MSO supplementedwith 50 mg of kanamycin per liter) for selection. Regenerated plantswill remain on this media for 2-4 weeks under 16 h/8 h light/dark and26° C. incubation.

When roots are sufficiently developed (2-5 roots, 2 cm or more inlength), plantlets are moved to ⅓ Half Hogland solution (Sigma ChemicalCo., St. Louis, Mo.) for 2-3 weeks to harden them prior to moving intosoil.

Transient β-Glucuronidase (GUS) expression is determined 5 days aftertransformation. The intact explants or regenerated shoots are subjectedto GUS histochemical assay (Jefferson et al., 1987, EMBO J.16:2901-2907). Transformation events are dark blue spots.

Radiolabeling of the cDNA or partial genomic clones for Ara h1 and Arah2 is performed using random oligonucleotides labeling (Amersham,Arlington Heights, Ill.) with ³²P-dCTP. The labeled probes are used forthe detection of stable integration of Ara h transgenes into transgenicplants.

Non-transformed controls is analyzed to determine basal levels of eachgene in transgenic peanuts. The differences in allergen level ofexpression between the controls and the transformed peanut plants helpin determining the level of downregulation in the transformed plants.

Copy number of transformants is also determined using Southern analysisas described in (Sambrook et al., supra). Great variability in the levelof gene expression between individual transgenic plant containing thesame introduced gene has been reported Rosahl et al., 1987. EMBO J.6:1155-1159. This variability has been ascribed to various factorsincluding gene copy number. A high correlation has been observed betweengene copy number and increased gene expression.

An equal amount of digested (XbaI/SacI) and undigested (intact) genomicDNA (10 μg per lane) is separated by agarose gel electrophoresis using a0.8% agarose gel, blotted onto a Hybond N⁺ membrane (NEN Life Sciences,Boston Mass.). Hybridization probes were the synthetic 78 nucleotidesDNA fragment for Ara h1 and the 80 nucleotides DNA fragment for Ara h2.Prehybridization, and hybridization is held at 60° C. for 2 hours andovernight, respectively. The membrane is washed twice for five minuteswith 2×SSC (1×SSC is 0.15 M NaCl plus 0.15 M sodium citrate), 0.1%sodium dodecyl sulfate (SDS) and twice with 0.2×SSC, 0.2% SDS at 60° C.for 15 min. Detection of hybridization patterns is performed byautoradiography. The hybridization pattern is used to determine the copynumber of the allergen genes per genome.

Enzyme-linked-immuno-sorbant-assay (ELISA) is performed to detectallergen levels in transgenic peanut plants. Proteins are extracted in aneutral pH phosphate buffer and ELISA conducted as described by Ausubelet al., 1995. In: Short Protocols in Molecular Biology. There arecurrently two commercial Elisa kits on the market for the detection ofpeanut residues: Neogen Corp. (Lansing, Mich.) and Elisa Technologies(Aluchua, Flor.)

Stable transformed peanut plants having undetectable or reduced orundetectable levels of peanut allergens are selected. Due to the use ofBiolistics in the transformation of peanut, multiple copies of each geneare found in multiple locations of the genome, resulting in enhanceddown-regulation.

Example 5 Verification of Transgene Transcripts, and Level ofDown-Regulation of Peanut Allergens

Radio-labeled probes, with 32P-dCTP of DNA sequences corresponding tothe transgenes cloned into the transformation vectors, are used insouthern and northern blots to detect stable transformations, copynumber of transgenes, and RNA transcripts.

An equal amount of digested (XbaI/SacI) and undigested (intact) genomicDNA (10 μg per line) is separated by agarose gel electrophoresis using a1% agarose gel, blotted onto a Hybond N⁺ membrane (NEN Life Sciences,Boston Mass.). Prehybridization, and hybridization are held at 60° C.for 2 hours and overnight, respectively. The membrane is washed twicefor five min with 2×SSC (1×SSC is 0.15 M NaCl plus 0.15M sodiumcitrate), 0.1% sodium dodecyl sulfate (SDS), and twice with 0.2×SSC,0.2% SDS at 60° C. for 15 min. Detection of hybridization patterns isperformed by autoradiography.

Enzyme-linked-immuno-sorbant-assay (ELISA) is performed to detectallergen levels in transgenic peanut plants. Proteins are extracted in aneutral pH phosphate buffer and ELISA conducted as described by Ausubelet al., 1995. In: Short Protocols in Molecular Biology. There arecurrently two commercial ELISA kits on the market for the detection ofpeanut residues: Neogen Corp. (Lansing, Mich.) and ELISA Technology(Aluchua, Flor.) Stable transformed peanut plants having undetectable orreduced levels of allergens are selected.

1. A method for producing a transgenic peanut plant with reduced orundetectable allergen protein content in the seed, comprising the stepsof: (a) transforming a recipient peanut plant cell with a DNA constructcomprising a peanut allergen antisense gene, or a peanut allergen sensegene, or a combination thereof, or fragments thereof; (b) regenerating apeanut plant from the recipient cell which has been transformed with theDNA construct; and (c) identifying a fertile transgenic peanut thatproduces seeds having reduced or undetectable allergen protein content.2. The method of claim 1, wherein the peanut allergen gene is selectedfrom the group consisting of Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Arah6, and Ara h7.
 3. The method of claim 1, wherein the recipient cell istransformed by the Agrobacterium-mediated method.
 4. The method of claim1, wherein the recipient cell is transformed by the biolistic method. 5.The method according to claim 1, wherein the peanut allergen sense orantisense gene, or a fragment thereof, comprises at least a portion ofthe nucleotide sequence shown in FIG. 2 (SEQ ID NO: 1).
 6. The methodaccording to claim 1, wherein the peanut allergen sense or antisensegene, or fragment thereof, comprises at least a portion of thenucleotide sequence shown in FIG. 3 (SEQ ID NO: 3).
 7. The methodaccording to claim 1, wherein the peanut allergen sense or antisensegene or fragment thereof, comprises at least a portion of the nucleotidesequence shown in FIG. 4 (SEQ ID NO: 4).
 8. The method according toclaim 1, wherein the peanut allergen sense or antisense gene, orfragment thereof, comprises at least a portion of the nucleotidesequence shown in (SEQ ID NO: 5)
 9. The method according to claim 1,wherein the peanut allergen sense or antisense gene, or fragmentthereof, comprises at least a portion of the nucleotide sequence shownin FIG. 7 (SEQ ID NO: 6).
 10. An isolated polynucleotide moleculecomprising the peanut allergen antisense gene, or fragment thereof,operably linked to a promoter and a terminator, the promoter andterminator functioning in a peanut cell.
 11. The polynucleotide moleculeof claim 10, wherein the antisense gene codes for an RNA molecule thatis complementary to the mRNA molecule coded for by a peanut allergenprotein gene selected from the group consisting of Ara h1, Ara h2, Arah3, Ara h4, Ara h5, Ara h6 and Ara h7.
 12. The polynucleotide moleculeaccording to claim 11, wherein the antisense gene has the nucleotidesequence selected from the group consisting of the nucleotide sequencesshown in FIGS. 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ ID NO: 5) and7 (SEQ ID NO: 6).
 13. The polynucleotide molecule according to claim 10,wherein the promoter is selected from the group consisting ofconstitutive, inducible and tissue-preferred promoter.
 14. Thepolynucleotide molecule according to claim 13, wherein the promoter is aseed-preferred promoter.
 15. A vector comprising the polynucleotidemolecule of claim
 10. 16. A peanut plant cell comprising thepolynucleotide molecule of claim
 10. 17. A peanut plant comprising thecell of claim
 16. 18. A seed produced by the plant of claim
 17. 19. Anisolated polynucleotide comprising the promoter of the Ara h2 genehaving the nucleotide sequence shown in FIG. 9 (residues 1-154 of SEQ IDNO: 1).
 20. An isolated polynucleotide consisting essentially of thenucleotide sequence selected from the group consisting of the nucleotidesequences shown in FIGS. 3 (SEQ ID NO: 3), 4 (SEQ ID NO: 4), 5 (SEQ IDNO: 5) and 7 (SEQ ID NO: 6).